Revision as of 17:54, 23 October 2017

labor:cell_culture - iGEM 2017

Lab Notebook Modeling

06.06.17

Titration RPMI 1640 vs. lactic acid

Aim: setup of pH in the Medium for pCRE-tests

stock-solutions:
c(lactic acid, conc.) = 12.09 mol/l
c(lactic acid, 1:20) = 0.6 mol/l (diluted with RPMI 1640)

Execution:

add lactic acid (0.6 M) to medium (Table)
measure pH with pH-Meter
repeat each measurement 3 times

V(RPMI 1640) [µl]	V(lactic acid, 0.6 M) [µl]	c(lactic acid) [µmol/mL]	s(c ) [µmol/ml]	pH 1	pH 2	pH 3	pH	s(pH)
2000	20	5.99	0.2	7.14	7.18	7.16	7.16	0.01
2000	40	11.9	0.3	6.90	6.91	6.88	6.90	0.01
2000	60	17.6	0.5	6.57	6.61	6.66	6.61	0.03
2000	80	23.3	0.6	6.35	6.31	6.27	6.31	0.02
2000	100	28.8	0.8	5.91	5.65	5.75	5.77	0.08
2000	120	34.2	0.9	4.99	4.96	4.93	4.96	0.02
2000	140	40	1	4.41	4.37	4.36	4.38	0.02
2000	160	45	1	4.31	4.12	4.10	4.18	0.07
2000	180	50	1	3.98	3.95	3.98	3.97	0.01

Resulting Titration curve for lactic acid in RPMI 1640:

Titration curve with pH vs. c(lactic acid)	Titration curve with pH vs. V(lactic acid 0.6 M) per ml

13.06.17

Cell survival and GFP-stability at different pH-values

used cells: Jurkat with CMV_GFP
cell density: 1 mio cells/ml
incubation times: 3 h, 6 h, 12 h

Execution:

count cells
centrifuge cells
resuspend cell pellet in RPMI 1640 (target-concentration: 2 mio cells/ml)
mix 1.5 ml RPMI 1640 + calculated volume of lactic acid (0.6 M) + 500 µl cell suspension on 12-well-plate (Table 1)
incubate for defined time in the CO₂-incubator
centrifuge samples
resuspend cell pellets in PBS FACS buffer (500 µl)
FACS analysis
used medium was stored at 4°C for pH-test next day

pH	V(lactic acid, 0.6 M) [µl]	RPMI 1640 [µl]
5.0	120	2000
5.5	107	2000
6.0	92	2000
6.5	68	2000
7.0	0	2000

Results:

→ Jurkat cells can survive in medium with pH ≥ 6.0

cell survival

after 3h	after 6h	after 12h

GFP fluorescence

after 3h	after 6h	after 12h

14.06.17

pH-measurement of used medium (13.06.17)

set pH	6 h	12 h
pH = 5.0	4.67	4.78
pH = 5.5	5.19	5.43
pH = 6.0	6.26	6.33
pH = 6.5	6.95	6.76
untreated (pH = 7.25)	7.73	7.47

Result: pH in incubation medium is rising over the time! Expected was a lowering, as the cells secrete lactic acid.

17.06.17

Investigation of the pH-changes

test of pH-change of RPMI 1640 at different conditions

tested conditions:

incubation in stock RPMI 1640 or RPMI 1640 with lactic acid (pH = 6.0)
medium with/without Jurkat cells (500k/ml)
incubation at 37°C in incubator with CO₂ (5%) atmosphere or incubated with cap
incubation at atmospheric conditions
incubation time: 6 h

make aliquots of RPMI 1640 (15 ml, pH = 6.0) and untreated RPMI 1640 (15 ml)
aliquot 1: measure pH before incubation
other aliquots: incubate at conditions described above

Results

6 h, 37 °C, 5% CO₂	RPMI 1640 (pH = 6.0)	stock RPMI 1640
pH before incubation	5.87	7.45
pH after incubation	6.46	7.68

6 h, 37 °C, 5% CO₂, Jurkat cells	RPMI 1640 (pH = 6.0)	stock RPMI 1640
pH before incubation	5.87	7.45
pH after incubation	6.48	7.70

6 h, 37 °C, with cap	RPMI 1640 (pH = 6.0)	stock RPMI 1640
pH before incubation	5.87	7.45
pH after incubation	6.63	8.46

6 h, 25 °C, atmospheric conditions	RPMI 1640 (pH = 6.0)	stock RPMI 1640
pH before incubation	5.87	7.45
pH after incubation	6.24	7.79

19.06.17

pH change vs. incubation time

set pH to 6.3
prepare 2 ml aliquots of RPMI 1640 in falcon tubes
incubate in CO₂ incubator for t (min) = 0, 10, 20, 30, 50
measure pH directly after taking sample out of incubator
measure pH one minute later

time /min	pH direct	pH after 1 min
0	6.32	6.32
10	6.03	6.25
20	6.06	6.28
30	5.88	6.36
50	6.36	6.65

pH is strongly rising after taking the sample out of the incubator. This may be caused by the solved CO₂, that lowers the pH in the incubator. After taking the sample out of the incubator, the CO₂ goes to the gas phase which causes a rising of the pH. Additionally, the bicarbonate buffer is destabilized as the addition of lactic acid moves the equilibrium of the buffer to CO₂ + H₂O. That leads to a higher pH than before the incubation.

04.07.17

VEGF dilution

m₀ = 10 µg start concentration: solid state target concentration: 1 µg/ml

resuspend pellet in 100 µl ddH₂O (stock solution, 100 µg/ml)
dilute 1 µl of stock solution with 99 µl ddH₂O (aliquots, 1 µg/ml)
store aliquots at -20 °C

06.07.17

pCTLA4 vs. [VEGF]

Aim: determination of suitable [VEGF] and incubation time

tested cells: HEK with transient pCTLA4-GFP (pIG17_022 (330 bp); pIG17_023 (380 bp)),PEI transfected
[VEGF](ng/ml) = 0.0, 0.5, 5.0, 10.0
t (h) = 3, 8, 24
controls: CMV_GFP (pIG_009), untransfected cells

Execution:

cells were PEI transfected by cell culture on 05.07.17
24h after PEI transfection: replace old medium by RPMI 1640 (2 ml / well), add VEGF (1 µg/ml)
after incubation time: analysis via fluorescence microscope and FACS

Results:

18.07.17

PEI transfection of plasmids pIG17_022/23/37/86

used cells: HEK cells

Plasmid number	component
pIG17_022	Ctla4(380bp)-GFP
pIG17_023	Ctla4(330bp)-GFP
pIG17_037	Ctla4(330bp)-GFP-CMV-mCherry
pIG17_086	Ctla4(380bp)-GFP-CMV-mCherry

Split the cells 1:5 into 10 cm plate (on 21.04.17)
Thaw PEI reagent at RT
Dilute plasmid in 1 ml serum free DMEM (2.5 µg from each plasmid)
Add 15 µl PEI(1 µg/µl) to the mix while vortexing (PEI:DNA = 3:1)
incubate 15 min at RT
Add the mix to the culture
incubate for 24 h

19.07.17

VEGF test

used cells: PEI transfected cells from 19.07.17 with pIG17_022/023/037/086

add following VEGF concentrations to the cells: 0 ng/ml, 0.5 ng/ml, 5 ng/ml, 10 ng/ml, 50 ng/ml
incubate for 24 h

fluorescence microscope:

construct	bright field	GFP
positive control CMV-CFP
negative control

05.08.17

Interlab Study transformation

- heat shock transformation of DH5-α with plasmids from Kit plate 7

well	approach
21B	positive control
21D	negative control
21F	Device 1
21H	Device 2
21J	Device 3
21L	Device 4
21N	Device 5
21P	Device 6

Interlab Study OD600 calibration measurements

- measure absorbance at 600 nm of H20 (100 µl) and LUDOX-S40 (100 µl) - each four replicates

replicate	LUDOX-S40	H2O
1	0,043	0,035
2	0,049	0,036
3	0,047	0,035
4	0,052	0,035

Interlab Study fluorescein standard curve measurements

- centrifuge fluorescein
- dilute fluorescein in PBS to a final concentration of 50 µM
- add PBS (100 µl) into well A2-A12, B2-B12, C2-C12, D2-D12 of a 96-well-plate
- add fluorescein stock solution (200 µl, 50µM) into well A1, B1, C1, D1
- transfer 100 µl from well A1 into well A2, pipett up and down
- transfer 100 µl from well A2 into well A3, pipett up and down
- do the same till the end of the row
- transfer 100 µl from every last well into the liquid waste
- repeat these steps for rpw B, C and D
- measure the fluorescence intensity in the plate reader

uM Fluorescein	50,00	25	12,5	6,25	3,125	1,5625	0,78125	0,390625	0,1953125	0,09765625	0,048828125	0
Replicate 1	41550	28587	15976	9537	5674	2712	1357	639	348	160	91	0
Replicate 2	42434	29060	17592	10169	5250	2705	1155	581	302	160	67	11
Replicate 3	42066	29363	17743	9882	5015	2671	1303	665	314	187	87	0
Replicate 4	40538	28766	17808	9934	5127	2818	1344	662	366	176	113	13

06.08.17

pick colonies from Interlab Study

- pick 2 colonies from each plate
- incubation overnight at 37°C

07.08.17

Interlab Study cell measurements

- measure OD600 of the overnight cultures
- dilute the bacteria in medium with Chloramphenicol to a final OD = 0.02
- incubation at 37°C and 220 rpm
- take 500 µl of the cultures after 0h, 2h, 4h and 6h and place samples on ice
- transfer four replicates of 100 µl of each sample into a well of a 96-well-plate
- measure OD600 and fluorescence intensity in the plate reader (settings: excitation: 485 nm; emission: 530/30)

Abs600 after 0h:

	Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6	LB + Chlor (blank)
Colony 1, Replicate 1	0,044	0,042	0,045	0,045	0,046	0,045	0,052	0,043	0,041
Colony 1, Replicate 2	0,046	0,047	0,045	0,045	0,047	0,048	0,046	0,064	0,041
Colony 1, Replicate 3	0,044	0,045	0,047	0,044	0,054	0,046	0,045	0,045	0,043
Colony 1, Replicate 4	0,045	0,045	0,045	0,058	0,045	0,046	0,044	0,046	0,046
Colony 2, Replicate 1	0,046	0,047	0,047	0,05	0,046	0,044	0,044	0,044	0,043
Colony 2, Replicate 2	0,046	0,05	0,045	0,045	0,045	0,046	0,05	0,046	0,046
Colony 2, Replicate 3	0,049	0,049	0,046	0,045	0,053	0,046	0,047	0,047	0,044
Colony 2, Replicate 4	0,046	0,051	0,043	0,043	0,044	0,049	0,048	0,05	0,042

Fluorescence intensity after 0h:

	Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6	LB + Chlor (blank)
Colony 1, Replicate 1	30032	29468	47522	30425	37202	30859	38967	39848	40167
Colony 1, Replicate 2	38825	32134	33823	37326	38129	41631	37103	40926	383
Colony 1, Replicate 3	33426	38291	40791	38829	35103	35503	39082	38194	40506
Colony 1, Replicate 4	30451	31664	40586	40661	37774	40046	38716	42854	39830
Colony 2, Replicate 1	31429	32597	33648	34085	40700	31595	28914	36532	40434
Colony 2, Replicate 2	39464	32361	39432	40150	39543	41282	38247	33320	39231
Colony 2, Replicate 3	32329	31485	34366	36754	39980	41296	33348	33391	39612
Colony 2, Replicate 4	31225	36435	37063	38806	42787	40222	36968	36323	38492

Abs600 after 2h:

	Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6	LB + Chlor (blank)
Colony 1, Replicate 1	0,046	0,045	0,045	0,065	0,047	0,047	0,046	0,044	0,041
Colony 1, Replicate 2	0,047	0,047	0,046	0,048	0,048	0,046	0,047	0,046	0,043
Colony 1, Replicate 3	0,046	0,047	0,046	0,045	0,053	0,047	0,048	0,045	0,044
Colony 1, Replicate 4	0,047	0,047	0,055	0,058	0,047	0,046	0,045	0,046	0,043
Colony 2, Replicate 1	0,078	0,048	0,048	0,049	0,048	0,047	0,046	0,047	0,043
Colony 2, Replicate 2	0,048	0,055	0,048	0,049	0,047	0,046	0,049	0,049	0,041
Colony 2, Replicate 3	0,047	0,048	0,048	0,048	0,047	0,049	0,048	0,05	0,045
Colony 2, Replicate 4	0,046	0,049	0,047	0,046	0,045	0,048	0,048	0,049	0,047

Fluorescence intensity after 2h:

	Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6	LB + Chlor (blank)
Colony 1, Replicate 1	32478	41305	33618	42435	34249	34205	34202	34949	29069
Colony 1, Replicate 2	34345	42706	38570	43860	36547	44267	45267	47187	37138
Colony 1, Replicate 3	38418	39069	38866	44974	36631	43986	46760	39103	39152
Colony 1, Replicate 4	36379	42391	36129	45361	39003	44229	32123	33637	41422
Colony 2, Replicate 1	36339	36658	36464	35639	43270	42121	34385	44627	40854
Colony 2, Replicate 2	40618	43596	36428	44147	44768	43257	45151	46402	39718
Colony 2, Replicate 3	39505	36517	43094	44747	36790	40034	44931	40597	39712
Colony 2, Replicate 4	36751	41857	35023	41187	35201	43423	32927	35063	37786

Abs600 after 4h:

	Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6	LB + Chlor (blank)
Colony 1, Replicate 1	0,056	0,055	0,055	0,052	0,058	0,052	0,053	0,046	0,042
Colony 1, Replicate 2	0,054	0,056	0,054	0,052	0,055	0,054	0,055	0,048	0,041
Colony 1, Replicate 3	0,055	0,061	0,055	0,052	0,057	0,052	0,055	0,049	0,043
Colony 1, Replicate 4	0,055	0,056	0,05	0,053	0,053	0,052	0,05	0,049	0,043
Colony 2, Replicate 1	0,053	0,059	0,057	0,061	0,053	0,059	0,053	0,059	0,043
Colony 2, Replicate 2	0,054	0,056	0,058	0,062	0,053	0,059	0,055	0,059	0,043
Colony 2, Replicate 3	0,054	0,056	0,058	0,063	0,057	0,059	0,055	0,06	0,043
Colony 2, Replicate 4	0,054	0,056	0,056	0,058	0,049	0,058	0,056	0,061	0,042

Fluorescence intensity after 4h:

	Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6	LB + Chlor (blank)
Colony 1, Replicate 1	39398	41211	43472	34629	40024	37129	34265	40517	38102
Colony 1, Replicate 2	39957	41499	44252	43313	44778	36906	43865	42985	38644
Colony 1, Replicate 3	39198	41382	35548	45574	47239	39866	44132	44431	37943
Colony 1, Replicate 4	35352	41590	26466	44768	45637	44736	29248	37825	38618
Colony 2, Replicate 1	41468	42577	34717	39284	40334	42558	46026	41271	39053
Colony 2, Replicate 2	43892	40143	36619	42028	41878	43940	38303	47567	37978
Colony 2, Replicate 3	43598	43486	44578	44449	44828	44235	42442	37273	39116
Colony 2, Replicate 4	43048	43193	42421	38871	34908	41518	43876	44582	37838

Abs600 after 6h:

	Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6	LB + Chlor (blank)
Colony 1, Replicate 1	0,098	0,093	0,084	0,084	0,087	0,078	0,083	0,062	0,048
Colony 1, Replicate 2	0,09	0,094	0,082	0,076	0,089	0,083	0,091	0,064	0,041
Colony 1, Replicate 3	0,091	0,09	0,92	0,082	0,089	0,08	0,086	0,066	0,043
Colony 1, Replicate 4	0,086	0,084	0,09	0,079	0,086	0,078	0,084	0,063	0,043
Colony 2, Replicate 1	0,081	0,093	0,094	0,076	0,075	0,097	0,087	0,097	0,043
Colony 2, Replicate 2	0,079	0,092	0,091	0,099	0,073	0,095	0,085	0,107	0,043
Colony 2, Replicate 3	0,08	0,086	0,101	0,13	0,073	0,1	0,088	0,107	0,042
Colony 2, Replicate 4	0,078	0,092	0,089	0,108	0,073	0,093	0,09	0,105	0,042

Fluorescence intensity after 6h:

	Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6	LB + Chlor (blank)
Colony 1, Replicate 1	44819	43197	34990	39952	35312	44026	44108	34395	41891
Colony 1, Replicate 2	35745	41624	42712	36773	38191	39327	36222	38684	44486
Colony 1, Replicate 3	43503	43470	38934	39338	39338	47418	46563	41520	40216
Colony 1, Replicate 4	40336	39507	37827	41517	41517	46271	47662	42298	38353
Colony 2, Replicate 1	34982	34672	46394	18665	18665	36165	36374	40293	40211
Colony 2, Replicate 2	43791	37273	44467	41079	41079	41516	44327	38827	38014
Colony 2, Replicate 3	39248	40869	38169	48933	48933	44903	41210	38643	40437
Colony 2, Replicate 4	38078	38846	41889	49892	49892	42376	46132	38525	39913

19.08.-27.08.17

pH change over time in incubator with RPMI1640 and acid

composition	start pH	incubation time
2ml RPMI1640	7.14	93 min
2ml RPMI1640	7.16	67 min
2ml RPMI1640	7.15	104 min
2ml RPMI1640 + 65µl lactic acid (0.6 M)	6.2	95 min
2ml RPMI1649 + 65µl lactic acid (0.6 M)	6.3	90 min
2ml RPMI1640 + 20µl HCl(1.2 M)	6.6	105 min
2ml RPMI1640 + 30µl HCl(1.2 M)	6.6	100 min
7.5 ml RPMI1640 + 250.000 HEK cells / ml	7.3	24 h
7.5 ml RPMI1640 + 500.000 HEK cells / ml	7.17	26 h

10.08.17

Transformation of pWHE-644

- transformation in DH5-α

12.08.17

Miniprep of pWHE-644 in DH3-α

- miniprep with Zymo kit

Test digest of pWHE-644

1µl NcoI
2µl DNA
1.5µl FD-buffer 10x
10.5µl H20
Excepted bands could be observed.

13.08.17

Miniprep of CMV-VEGFR2 (pIG_138)

- 7x miniprep of CMV-VEGFR2

Test digest of CMV-VEGFR2 (pIG_138)

2µl DNA
1.5µl buffer
1µl BamHI
0.5µl H20
Only one of the expected bands could be oberserved.

13.08.17

Test digest of CMV-VEGFR2 (pIG_138)

2µl DNA
1.5µl FD-buffer
1µl SmaI
Only one of the expected bands could be oberserved.

16.08.17

pH test with Jurkat cells

- centrifuge Jurkatt cells, dilute in RPMI1640
- count cells
- incubation of Jurkat cells and lactic acid
- pH measurements after 0h, 30min, 1h and 1h30min
- 1) only RPMI1640 with cells, start pH at 7.2
- 2) RPMI1640 with cells and 100µl lactic acid, start pH at 6.8

after 0h:

cell concentration (cells/ml)	pH of 1)	pH of 2)
3mio	7.46	6.81
2mio	7.41	6.82
1.5mio	7.47	6.86
1mio	7.49	6.79
500k	7.45	6.78
100k	7.45	6.77

after 30min:

cell concentration (cells/ml)	pH of 1)	pH of 2)
3mio	7.52	7.18
2mio	7.51	7.13
1.5mio	7.53	7.14
1mio	7.52	7.11
500k	7.54	7.07
100k	7.55	7.12

after 1h:

cell concentration (cells/ml)	pH of 1)	pH of 2)
3mio	7.48	7.26
2mio	7.57	7.22
1.5mio	7.60	7.24
1mio	7.60	7.22
500k	7.60	7.18
100k	7.63	7.21

after 1h30min:

cell concentration (cells/ml)	pH of 1)	pH of 2)
3mio	7.42	7.14
2mio	7.55	7.27
1.5mio	7.46	7.35
1mio	7.62	7.18
500k	7.51	7.23
100k	7.59	7.26

16.08.17

Test digest of CMV-VEGFR2 (pIG_138)

2µl DNA
1.5µl FD-buffer
2µl BamHI
9,5µl H20

Ctla4 induction

- Jurkat cells transiently transfected with Ctla4-GFP-CMV-mCherry
- positive control: cotransfected with CMV-GFP and CMV mCherry
- add VEGF
- incubation for 24h at 37°C

17.08.17

PEI transfection of HEK cells

- CMV-GFP from iGEM
- CMV-GFP from tool box

Flow cytometry

- Jurkat with Ctla4-GFP-CMV-mCherry from 16.08.17

mCherry	GFP (measured in green fluorescence channel)

18.08.17

BioRad electroporation

Cell lines: Jurkat
Plasmids: pIG_037, pIG_031, contransfection CMV-GFP

Count the cells
Transfer 1 mio. cells into falcons and centrifuge with 100g for 3 min
Resuspend the cell pellet with 100 µl P3 Buffer
Add 9 µg pIG17_009
Transfer the mixture into cuvette
BioRad setting: 250 V, 960 µF, 35 sec.
Add 500 µl RPMI medium to the cells immediately after electroporation
Plate the cells into 12-well plate with 2 ml Medium
Over night culturing
Wash the cuvettes for reusing

Calcium test

- incubation of cells at 37°C in FACS tubes for 5min - pre-FACS after 3min - add VEGF (30ng/ml, 70ng/ml) - FACS afer 7min: no signal

20.08.17

Biorad electroporation

Cell lines: Jurkat
Plasmids: pIG_034, cotransfection CMV-GFP and CMV-mCherry, negative control (no DNA)

Count the cells
Transfer 1 mio. cells into falcons and centrifuge with 100g for 3 min
Resuspend the cell pellet with 100 µl P3 Buffer
Add 6 µg pIG17_009
Transfer the mixture into cuvette
BioRad setting: 250 V, 960 µF, 35 sec.
Add 500 µl RPMI medium to the cells immediately after electroporation
Plate the cells into 12-well plate with 2 ml Medium
Over night culturing
Wash the cuvettes for reusing

pH tests with Jurkat

V(ges) [ml]	V(RPMI1640) [ml]	V(cells) [ml]	c(cells) [cells/ml]
7.5	5.3	2.2	250.000

- incubation for 24h

Miniprep of pIG_034

- miniprep 8x

21.08.17

sequencing of pIG_138

- plasmid: CMV-VEGFR2 (pIG_138)
- DNA concentration: 80ng/ml
- Oligo: 2µl DNA, 18µl H2O
- V(ges): 20µl

22.08.17

Miniprep of pIG_017, pIG_031, pIG_034, pIG_037

- Zymo kit

up-concentration of pIG_017, pIG_031, pIG_034, pIG_037

- add isopropanol to plasmid suspension in H2O
- -20°C for 20min
- centrifugate for 30min at maximal speed and 4°C
- remove isopropanol
- wash with EtOH (70%)
- centrifugate for 30min att maximal speed and 4°C
- remove EtOH
- open eppis to dry overnight
- eluate with H2O
Up-conentration did not work, DNA concentration decreased.

25.08.17

Miniprep of pIG_017, pIG_037

07.09.17

PEI transfection of HEK cells

used cells: HEK cells

Plasmid number	component
pIG17_034	pCRE-GFP-CMV-mCherry
pIG17_008 + pIG17_009	CMV-GFP + CMV-mCherry
pIG17_034 + β2-receptor	pCRE-GFP-CMV-mCherry + β2-receptor
no DNA	negative control

Split the cells 1:5 into 10 cm plate
Thaw PEI reagent at RT
Dilute plasmid in 1 ml serum free DMEM (2.5 µg from each plasmid)
Add 15 µl PEI(1 µg/µl) to the mix while vortexing (PEI:DNA = 3:1)
incubate 15 min at RT
Add the mix to the culture
incubate for 24 h

Gel extraction of M1-receptor without CFP

50 ng DNA
1 µl T4 Ligase
2 µl Buffer 10x
13 µl H2O

08.09.17

Colony PCR of M1

Buffer 10x	2.5µl	12.5 µl
dNTP 10 mM	0.5 µl	2.5 µl
Template	1 µl
Primer	0.5 µl	2.5 µl
DreamTaq Thermo Fisher	0.25 µl	1.25 µl
H2O	20 µl	100 µl

20.08.17

pCRE induction

- treatment in 6-well-plate
- add Forskolin and β2-inductor to the cells

Cells transfected with pIG17_034 (pCRE-GFP-CMV-mCherry):

approach	Forskolin	β2-inductor
negative control	-	-
old Forskolin	10 µM (old)	-
new Forskolin	10 µM (new)	-
new Forskolin	100 µM new Forskolin
β2-inductor	-	10 µM
β2-inductor	-	100 µM

Cells transfected with pIG17_034 (pCRE-GFP-CMV-mCherry) and β2-receptor:

approach	Forskolin	β2-inductor
negative control	-	-
β2-inductor	-	10 µM
β2-inductor	-	100 µM

10.09.17

PEI transfection of plasmids pIG17_022/23/37/86

used cells: HEK cells

Plasmid number	component
pIG17_031	HRE-GFP-CMV-mCherry

Split the cells 1:2 into 10 cm plate
Thaw PEI reagent at RT
Dilute plasmid in 1 ml serum free DMEM (2.5 µg from each plasmid)
Add 15 µl PEI(1 µg/µl) to the mix while vortexing (PEI:DNA = 3:1)
incubate 15 min at RT
Add the mix to the culture
incubate for 24 h

HRE induction

- add CoCl2 (end concentrations: 0 µM, 5 µM, 10 µM, 20 µM, 50 µM, 100 µM, 200 µM, 400 µM, 800 µM)
- fluoresence microscopy

Repetition of the Interlab Study

- transformation of DH5-α with plasmids from Kit 7 (21B, 21D, 21F, 21H, 21L, 21N, 21P)
- incubation at 37 °C

PEI transfection of HEK cells

Plasmid number	component
pIG17_034 + L3 (Luciferase)	pCRE-GFP-CMV-mCherry + L3
pIG17_034 + TDAG8	pCRE-GFP-CMV-mCherry + TDAG8
pIG17_008 + pIG17_009	CMV-GFP + CMV-mCherry

16.09.17

Pick colonies of Interlab Study

- 2 colonies per plate
- incubation at 37°C

pCRE induction in HEK cells

- plate out cells on 24-well-dish
- treatment with Forskolin

Plasmid number	component	Forskolin treatment
pIG17_008 + pIG17_009	CMV-GFP + CMV-mCherry	-
pIG17_034 + TDAG8	pCRE-GFP-CMV-mCherry + TDAG8	+
no DNA	-	-
no DNA	-	+
pIG17_034	pCRE-GFP-CMV-mCherry	-
pIG17_034	pCRE-GFP-CMV-mCherry	+

- after 10 hours: pH change to 6.5, 7.0, 7.5
- plate reader after 1h, 3h, 6h, 12h, 24h (Ex.: 488±20 nm; Emm.: 509±20 nm)
- FACS analysis after 24h

FACS

Plate Reader

17.09.17

PEI transfection of plasmid pIG17_031

used cells: HEK cells

Plasmid number	component
pIG17_031	HRE-GFP-CMV-mCherry
pIG17_008 + pIG17_009	CMV-GFP + CMV-mCherry

Split the cells 1:2 into 10 cm plate (on 16.09.17)
Thaw PEI reagent at RT
Dilute plasmid in 1 ml serum free DMEM (2.5 µg from each plasmid)
Add 15 µl PEI(1 µg/µl) to the mix while vortexing (PEI:DNA = 3:1)
incubate 15 min at RT
Add the mix to the culture
incubate for 24 h

18.09.17

HRE induction

- induction cancelled because cells were contaminated with bacteria

19.09.17

overnights of overnights (HRE4x, CRE4x)

20.09.17

Midiprep of cultures from overnights

PEI transfection

used cells: HEK cells

Plasmid number	component
+ TDAG8	CRE(4x)-GFP-CMV-mCherry + TDAG8
	HRE(4x)-GFP-CMV-mCherry
pIG17_008 + pIF17_009	CMV-GFP + CMV-mCherry

Split the cells 1:2, transfer into 6-well-plate (on 16.09.17)
Thaw PEI reagent at RT
Dilute plasmid in 1 ml serum free DMEM (2.5 µg from each plasmid)
Add 15 µl PEI(1 µg/µl) to the mix while vortexing (PEI:DNA = 3:1)
incubate 15 min at RT
Add the mix to the culture
incubate for 24 h

Filtration of DMEM, addition of FBS

- filter DMEM sterile
- add 2 % FBS
- set up pH of aliquots with HCl (4 M)
- final pH values: 6.7, 7.1, 7.4

22.09.17

induction of CRE and HRE in HEK cells

- indution in 96-wel-plates - cells transiently transfected with CRE-GFP-CMV-mCherry, add acid
- cells transiently transfected with HRE-GFP-CMV-mCherry, add CoCl2
- plate reader: no measurable induction of GFP

23.09.17

FACS analysis of HEK cells

- FACS analysis after CRE induction and HRE induction

CREx4_GFP	HREx4_GFP

24.09.17

Repetition of the Interlab Study

- transformation of DH5-α with plasmids from Kit 6 (20B, 20D, 20F, 20H, 20L, 20N, 20P)
- incubation at 37 °C

29.09.17

calibration of plate reader

- used cells: knockdown-cells + untransfected cells, compositions 1:0, 1:1, 1:2, 1:4, 0:1
- 96-well-plates, black, clear bottom, 100 000 cells per well
- different media: RPMI164, DMEM, DMEM and PBS (500 000 cells per well)

induction of HRE(4x)

- used cells: HEK stably transfected with HRE(4x)-GFP-CMV-mCherry, untransfected as a negative control
- induction in 24-well-plate
- add CoCl2: 0 µM, 20 µM, 40 µM, 80 µM, 100 µM, 320 µM, 640 µM
- FACS analysis

02.10.17

pH measurements with HEK cells

- 6-well-plate, 900 000 cells per well (90% confluency)
- DMEM (5 ml per well), pH set to 6.8
- after 6 h: pH=7.2

induction of HRE

- used cells: Jurkat and HEK, stably transfected with HRE(4x)-GFP-CMV-mCherry
- induction with CoCl2: 0 µM, 50 µM, 100 µM, 150 µM, 200 µM, 250 µM, 300 µM, 350 µM

pH measurements with HEK cells

- 6-well-plate, 900 000 cells per well
- DMEM (5 ml per well), pH set to 6.73

03.10.17

indcution of CRE(4x) in HEK cells

- HEK stably transfected with CRE-GFP-CMV-mCherry, induction with acid (pH 6.7, 7.4, 8.1)
- FACS measurement failed

FACS analysis of CoCl2 treated HEK cells

05.10.17

resuspended HEK cells containing stable CREx4 were diluted at different ratios with wildtype HEK cells were incubated at different pH values (6.5, 7.1, 7.7) and measured on a plate reader.
normalization via OD600

14.10.17

Repetition of the Interlab Study

- transformation of positive control, negative control, Device 1-6

15.10.17

pick colonies of the Interlab Study

17.10.17

calibration plate reader

-calibration of plate reader for CFP with dilutions of knockdown Jurkat and HEK cells (CMV_CFP)
-black 96 well plate with clear bottom
-cells/well: 50k, 100k
-wavelengths: Ex.: 433±20 nm; Emm.: 475±9 nm

HEK	Jurkat

cell measurements of the Interlab Study

- OD600 and fluorescence intensity of each device after 0h, 2h, 4h and 6h
- again random fluorescence values

PEI transfection of CMV-VEGFR2, SV40-TDAG8

plasmid
CMV-VEGFR2
SV40-TDAG8

Split the cells 1:2 into 10 cm plate
Thaw PEI reagent at RT
Dilute plasmid in 1 ml serum free DMEM (2.5 µg from each plasmid)
Add 15 µl PEI(1 µg/µl) to the mix while vortexing (PEI:DNA = 3:1)
incubate 15 min at RT
Add the mix to the culture
incubate for 24 h

18.10.17

input tests in HEK and Jurkat cells

used medium: PBS (10% FCS, 0.5% glucose, 1% HEPES)

fluorescence measurement after 0h, 1h, 2h, 4h, 6h, 9h, 16, 24h

in order to determine the relative fluorescence intensity (RFI) stable cell lines were substracted by wildtype cells with same treatment.

CRE(4x)-CFP-CMV-mCherry (stable), SV40-TDAG8 (transient):

conditions:

pH	Forskolin + IBMX (100 µM each)	transient SV40_TDAG8 (HEK only)
6.5	-	-
6.5	+	-
6.5	-	+
6.5	+	+
7.1	-	-
7.1	+	-
7.1	-	+
7.1	+	+
7.7	-	-
7.7	+	-
7.7	-	+
7.7	+	+
6.5 → 7.7	-	-
6.5 → 7.7	+	-
6.5 → 7.7	-	+
6.5 → 7.7	+	+

Results:

HEK293T	Jurkat

Ctla4(4x)-CFP-CMV-mCherry (stable), CMV-VEGFR-2 (transient):

conditions:

VEGF (ng/ml)	Ionomycin (µmol/l)	transient CMV-VEGFR-2 (HEK only)
0		+
0		-
25		+
25		-
50		+
50		-
100		+
100		-
	1.25	+
	1.25	-
	2.5	+
	2.5	-
	5.0	+
	5.0	-
	10.0	+
	10.0	-

Results:

HEK293T	Jurkat

HRE(4x)-CFP-CMV-mCherry (stable):

conditions:

CoCl2 (µmol/ml)
0
50
100
200
400
600

Results:

HEK293T	Jurkat
	high mCherry
	low mCherry

19.10.17

HRE, Ctla4 and HRE induction

- medium change for Ctla4 and HRE cells: PBS (10% FCS, 0.5% glucose, HEPES)
- medium change for CRE: PBS (10% FCS, 0.5% glucose, HEPES) pH = 7.7
- fluorescence measurement after 0h, 3h, 6h, 12h, 24h, 30h

@@ Line 91: / Line 91: @@
 <div class="container">
-    <div class="row">
+<div class="row">
-      <div class="col-md-12 text-center">
+<div class="col-md-12 text-center">
-        <div class="flex-container">
+<div class="flex-container">
-          <div class="item">
+<div class="item">
-<h1 class="sectionedit1" id="lab_notebook_suicide_gene_cloning">Lab Notebook Knockout</h1>
+    <h1 align="center">Lab Notebook Modeling</h1>
 <div class="level1">
 </div>
-<div class='secedit editbutton_section editbutton_1'> </div>
+              <button class="accordion">June</button>
+         <div class="panel panel-default">
-              <button class="accordion">May</button>
+<div class='secedit editbutton_section editbutton_1'> </div>
-        <div id="vanish" class="panel panel-default">
+<h2 class="sectionedit2" id="section060617">06.06.17</h2>
-<h2 class="sectionedit2" id="section06052017">06.05.2017</h2>
 <div class="level2">
 </div>
 <div class='secedit editbutton_section editbutton_2'> </div>
-<h3 class="sectionedit3" id="retransformation_of_lenti-cas9-puro_plasmid">Retransformation of lenti-CAS9-puro plasmid</h3>
+<h3 class="sectionedit3" id="titration_rpmi_1640_vs_lactic_acid">Titration RPMI 1640 vs. lactic acid</h3>
 <div class="level3">
 <p>
-A heatshock transformation of XL10 gold was performed with the lenti-CAS9-puro plasmid.<br/>
+<em class="u"><strong>Aim:</strong></em> setup of pH in the Medium for pCRE-tests<br/>
 </p>
-</div>
-<div class='secedit editbutton_section editbutton_3'> </div>
-<h2 class="sectionedit4" id="section07052017">07.05.2017</h2>
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_4'> </div>
-<h3 class="sectionedit5" id="producing_overnight_cultures">Producing overnight cultures</h3>
-<div class="level3">
 <p>
-Two colonies were observed. <br/>
+stock-solutions:<br/>
-ml LB-Medium containing the appropriate antibiotic was inoculated and incubated overnight at 37°C, 180rpm.<br/>
+c(lactic acid, conc.) = 12.09 mol/l<br/>
-</p>
+c(lactic acid, 1:20) = 0.6 mol/l (diluted with RPMI 1640)<br/>
-</div>
-<div class='secedit editbutton_section editbutton_5'> </div>
-<h3 class="sectionedit6" id="test_digest_of_lenti-cas9-puro">Test digest of lenti-CAS9-puro</h3>
-<div class="level3">
-<p>
-.66 μg DNA ( 3ul ~ 220ng/μl)<br/>
-.25 μl NcoI-HF<br/>
-μl CutSmart 10x buffer<br/>
-.75μl H20<br/>
 </p>
 <p>
-Digestion at 37°C for 1 h.<br/>
+<em class="u"><strong>Execution:</strong></em>
-→ Gel did not show separation of bands. Predicted fragments: ~6,100bp; 7,400bp<br/>
 </p>
+<ol>
-</div>
+<li class="level1"><div class="li"> add lactic acid (0.6 M) to medium (Table)</div>
-<div class='secedit editbutton_section editbutton_6'> </div>
+</li>
-<h2 class="sectionedit7" id="section08052017">08.05.2017</h2>
+<li class="level2"><div class="li"> measure pH with pH-Meter</div>
-<div class="level2">
+</li>
+<li class="level2"><div class="li"> repeat each measurement 3 times</div>
-</div>
+</li>
-<div class='secedit editbutton_section editbutton_7'> </div>
+</ol>
-<h3 class="sectionedit8" id="miniprep_of_lenti-cas9-puro">Miniprep of lenti-CAS9-puro</h3>
+<div class="table sectionedit4"><table class="inline">
-<div class="level3">
-<p>
-Plasmid was extracted using the provided protocol (Quiagen Kit).<br/>
-DNA was eluated in 30μl H2O.<br/>
-</p>
-</div>
-<div class='secedit editbutton_section editbutton_8'> </div>
-<h2 class="sectionedit9" id="section09052017">09.05.2017</h2>
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_9'> </div>
-<h3 class="sectionedit10" id="test_digest_of_lenti-cas9-puro1">Test digest of lenti-CAS9-puro</h3>
-<div class="level3">
-<p>
-μl Plasmid<br/>
-.25 μl NcoI-HF <br/>
-μl CutSmart 10x buffer <br/>
-.75 μl H20<br/>
-</p>
-<p>
-Digest was performed with lenti-CAS9-puro plasmids obtained from Minipreps and from the origial stock.<br/>
-Digestion at 37°C for 1h.<br/>
-</p>
-<p>
-Gel: 1.15h at 180V. Expected bands could be observed in all samples.<br/>
-</p>
-</div>
-<div class='secedit editbutton_section editbutton_10'> </div>
-<h2 class="sectionedit11" id="section1052017">10.5.2017</h2>
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_11'> </div>
-<h3 class="sectionedit12" id="test_digestion_of_cathomen_cas9_plasmid">Test digestion of Cathomen Cas9 plasmid</h3>
-<div class="level3">
-<p>
-<a href="/igem2017/lib/exe/detail.php?id=notebooks%3Aknockout&amp;media=notebooks:test_digestion_cas9_cathomen.png" class="media" title="notebooks:test_digestion_cas9_cathomen.png"><img src="/igem2017/lib/exe/fetch.php?w=400&amp;tok=9cc8d8&amp;media=notebooks:test_digestion_cas9_cathomen.png" class="media" alt="" width="400" /></a>
-</p>
-</div>
-<div class='secedit editbutton_section editbutton_12'> </div>
-<h2 class="sectionedit13" id="section11052017">11.05.2017</h2>
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_13'> </div>
-<h3 class="sectionedit14" id="retransformation_of_knockdown_plasmids">Retransformation of knockdown plasmids</h3>
-<div class="level3">
-<p>
-A heatshock transformation of XL10 gold was performed with the following plasmids: SDEN, SBEN, EDEN.<br/>
-Plasmids were provided by AG Schamel.<br/>
-</p>
-</div>
-<div class='secedit editbutton_section editbutton_14'> </div>
-<h2 class="sectionedit15" id="section12052017">12.05.2017</h2>
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_15'> </div>
-<h3 class="sectionedit16" id="producing_overnight_cultures_of_sden_sben_eden">Producing overnight cultures of SDEN, SBEN, EDEN</h3>
-<div class="level3">
-<p>
-ml LB-Medium containing the appropriate antibiotic was inoculated and incubated overnight at 37°C and 30°C; 180rpm.<br/>
-</p>
-</div>
-<div class='secedit editbutton_section editbutton_16'> </div>
-<h2 class="sectionedit17" id="section13052017">13.05.2017</h2>
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_17'> </div>
-<h3 class="sectionedit18" id="retransformation_of_cas9-gfp_plasmid">Retransformation of CAS9-GFP plasmid</h3>
-<div class="level3">
-<p>
-A heatshock transformation of XL10 gold was performed with the CAS9-GFP plasmid.<br/>
-</p>
-</div>
-<div class='secedit editbutton_section editbutton_18'> </div>
-<h3 class="sectionedit19" id="miniprep_of_sden_sben_eden">Miniprep of SDEN, SBEN, EDEN</h3>
-<div class="level3">
-<p>
-Plasmid was extracted using the provided protocol (Quiagen Kit).<br/>
-</p>
-<div class="table sectionedit20"><table class="inline">
 	<thead>
 	<tr class="row0">
-		<th class="col0">Temp.</th><th class="col1">Prep 1</th><th class="col2">Prep 2</th><th class="col3">Prep 3</th>
+		<th class="col0">V(RPMI 1640) [µl]</th><th class="col1">V(lactic acid, 0.6 M) [µl]</th><th class="col2">c(lactic acid) [µmol/mL]</th><th class="col3">s(c ) [µmol/ml]</th><th class="col4">pH 1</th><th class="col5">pH 2</th><th class="col6">pH 3</th><th class="col7">pH</th><th class="col8">s(pH)</th>
 	</tr>
 	</thead>
 	<tr class="row1">
-		<td class="col0">30°C</td><td class="col1">17,5 ng/μl</td><td class="col2">24,0ng/μl</td><td class="col3">34,0ng/μl</td>
+		<td class="col0">2000</td><td class="col1">20</td><td class="col2">5.99</td><td class="col3">0.2</td><td class="col4">7.14</td><td class="col5">7.18</td><td class="col6">7.16</td><td class="col7">7.16</td><td class="col8">0.01</td>
 	</tr>
 	<tr class="row2">
-		<td class="col0">37°C</td><td class="col1">53,1ng/μl</td><td class="col2">18,4g/μl</td><td class="col3">11,0ng/μl</td>
+		<td class="col0">2000</td><td class="col1">40</td><td class="col2">11.9</td><td class="col3">0.3</td><td class="col4">6.90</td><td class="col5">6.91</td><td class="col6">6.88</td><td class="col7">6.90</td><td class="col8">0.01</td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_20'> </div>
-</div>
-<div class='secedit editbutton_section editbutton_19'> </div>
-<h2 class="sectionedit21" id="section14052017">14.05.2017</h2>
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_21'> </div>
-<h3 class="sectionedit22" id="producing_overnight_cultures_cas9-gfp">Producing overnight cultures CAS9-GFP</h3>
-<div class="level3">
-<p>
-ml LB-Medium containing the appropriate antibiotic was inoculated and incubated overnight at 37°C; 180rpm.<br/>
-</p>
-</div>
-<div class='secedit editbutton_section editbutton_22'> </div>
-<h2 class="sectionedit23" id="section15052017">15.05.2017</h2>
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_23'> </div>
-<h3 class="sectionedit24" id="miniprep_of_cas9-gfp">Miniprep of CAS9-GFP</h3>
-<div class="level3">
-<p>
-Plasmid was extracted using the provided protocol (Quiagen Kit).<br/>
-Prep 1: 404.4 ng/μl<br/>
-Prep 2: 495.5 ng/μl<br/>
-</p>
-</div>
-<div class='secedit editbutton_section editbutton_24'> </div>
-<h3 class="sectionedit25" id="testdigest_of_cas9-gfp">Testdigest of CAS9-GFP</h3>
-<div class="level3">
-<div class="table sectionedit26"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0"> </th><th class="col1">origial stock</th><th class="col2">Prep 1</th><th class="col3">Prep 2</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0">DNA</td><td class="col1">0.25 μl</td><td class="col2">1 μl</td><td class="col3">1 μl</td>
-	</tr>
-	<tr class="row2">
-		<td class="col0">NcoI-HF</td><td class="col1">0.25 μl</td><td class="col2">0.25 μl</td><td class="col3">0.25 μl</td>
 	</tr>
 	<tr class="row3">
-		<td class="col0">CutSmart 10x</td><td class="col1">1 μl</td><td class="col2">1 μl</td><td class="col3">1 μl</td>
+		<td class="col0">2000</td><td class="col1">60</td><td class="col2">17.6</td><td class="col3">0.5</td><td class="col4">6.57</td><td class="col5">6.61</td><td class="col6">6.66</td><td class="col7">6.61</td><td class="col8">0.03</td>
 	</tr>
 	<tr class="row4">
-		<td class="col0">H2O</td><td class="col1">7.75 μl</td><td class="col2">6.75 μl</td><td class="col3">6.75 μl</td>
+		<td class="col0">2000</td><td class="col1">80</td><td class="col2">23.3</td><td class="col3">0.6</td><td class="col4">6.35</td><td class="col5">6.31</td><td class="col6">6.27</td><td class="col7">6.31</td><td class="col8">0.02</td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_26'> </div>
-<p>
-Digestion at 37°C for 1h.<br/>
-Expected fragments: ~500bp; 8,500bp.<br/>
-Expected fragments were observed in all samples.<br/>
-</p>
-</div>
-<div class='secedit editbutton_section editbutton_25'> </div>
-<h2 class="sectionedit27" id="section18052017">18.05.2017</h2>
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_27'> </div>
-<h3 class="sectionedit28" id="pcr_for_hif1a_exons">PCR for HIF1A exons</h3>
-<div class="level3">
-<p>
-μl DNA<br/>
-.25 μl Primer Fwd<br/>
-.25 μl Primer Rev<br/>
-.5 μl KAPA 2x<br/>
-</p>
-<p>
-As template gDNA from JK, HUT and HPB All cells was used. <br/>
-PCR program:<br/>
-min.  95°C<br/>
-sec. 95°C<br/>
-sec. 54°C<br/>
-sec. 72°C<br/>
-min. 72°C<br/>
-</p>
-<p>
-The expected bands were cut out of the gel and purified the next day; gel was stored at 4°C overnight.
-</p>
-</div>
-<div class='secedit editbutton_section editbutton_28'> </div>
-<h2 class="sectionedit29" id="section19052017">19.05.2017</h2>
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_29'> </div>
-<h3 class="sectionedit30" id="gel_extraction">Gel extraction</h3>
-<div class="level3">
-<p>
-Gel extraction was performed following the manufacturer&#039;s protocol (Quiagen Gel extraction kit).
-</p>
-<div class="table sectionedit31"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">Cell line</th><th class="col1">Exon 5</th><th class="col2">Exon 6</th><th class="col3">Exon 8</th><th class="col4">Exon 10</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0">JK</td><td class="col1">16.6 ng/μl</td><td class="col2">33.5 ng/μl</td><td class="col3">31.5 ng/μl</td><td class="col4">20.6 ng/μl</td>
-	</tr>
-	<tr class="row2">
-		<td class="col0">HPB</td><td class="col1">23.1 ng/μl</td><td class="col2">34.6 ng/μl</td><td class="col3">26.0 ng/μl</td><td class="col4">12.2 ng/μl</td>
-	</tr>
-	<tr class="row3">
-		<td class="col0">HUT</td><td class="col1">14.1 ng/μl</td><td class="col2">15.9 ng/μl</td><td class="col3">25.2 ng/μl</td><td class="col4">18.6 ng/μl</td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_31'> </div>
-</div>
-<div class='secedit editbutton_section editbutton_30'> </div>
-<h3 class="sectionedit32" id="retransformation_of_ko-kit_plasmids">Retransformation of KO-Kit plasmids</h3>
-<div class="level3">
-<p>
-A heatshock transformation of XL10 gold was performed with the plasmids 101,104,203.<br/>
-</p>
-</div>
-<div class='secedit editbutton_section editbutton_32'> </div>
-<h2 class="sectionedit33" id="section20052017">20.05.2017</h2>
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_33'> </div>
-<h3 class="sectionedit34" id="miniprep_and_midiprep_of_several_plasmids">Miniprep and Midiprep of several plasmids</h3>
-<div class="level3">
-<p>
-Plasmid was extracted using the provided protocol (Quiagen Miniprep Kit).<br/>
-The following plasmids were extracted: 203,104,101.<br/>
-</p>
-<p>
-Plasmid was extracted using the provided protocol (JetStar Midiprep Kit).<br/>
-The following plasmids were extracted: SDEN, SBEN, EDEN, lenti-CAS9-puro.<br/>
-</p>
-</div>
-<div class='secedit editbutton_section editbutton_34'> </div>
-<h2 class="sectionedit35" id="section23052017">23.05.2017</h2>
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_35'> </div>
-<h3 class="sectionedit36" id="digestion_of_cas9-gfp_by_bbsi">Digestion of CAS9-GFP by BbsI</h3>
-<div class="level3">
-<p>
-DNA from stock 2.<br/>
-μl DNA [0.5ng/μl]<br/>
-.5 μl BbsI<br/>
-μl 10x NEB2 buffer<br/>
-μl H2O<br/>
-</p>
-</div>
-<div class='secedit editbutton_section editbutton_36'> </div>
-<h3 class="sectionedit37" id="annealing_of_sgrna_oligos">Annealing of sgRNA oligos</h3>
-<div class="level3">
-<p>
-μl 100mM of each Fwd and Rev ssOligos<br/>
-μl 10x NEB2.1 buffer<br/>
-μl H2O<br/>
-min. at 95°C cooling down in heatblock for 2h.<br/>
-</p>
-</div>
-<div class='secedit editbutton_section editbutton_37'> </div>
-<h3 class="sectionedit38" id="testdigest_of_ko-kit_plasmids">Testdigest of KO-Kit plasmids</h3>
-<div class="level3">
-<p>
-Digest with EcoRV.<br/>
-.5 μl 10x Buffer<br/>
-μl DNA<br/>
-μl EcoRV<br/>
-,5 μl H2O<br/>
-(Gelpicture-number:299)<br/>
-</p>
-</div>
-<div class='secedit editbutton_section editbutton_38'> </div>
-<h3 class="sectionedit39" id="midiprep_of_sben_lenti-cas9-puro">Midiprep of SBEN, lenti-CAS9-puro</h3>
-<div class="level3">
-<p>
-Plasmid was extracted using the provided protocol (JetStar Midiprep Kit).<br/>
-The following plasmids were extracted: SBEN, lenti-CAS9-puro.<br/>
-</p>
-</div>
-<div class='secedit editbutton_section editbutton_39'> </div>
-<h2 class="sectionedit40" id="section250517">25.05.17</h2>
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_40'> </div>
-<h3 class="sectionedit41" id="testdigest_of_ko-kit_101_104_203_with_ecorv">Testdigest of KO-KIT (101,104,203) with EcoRV</h3>
-<div class="level3">
-<div class="table sectionedit42"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">Volume</th><th class="col1">Component</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0">1,5μl </td><td class="col1"> Buffer</td>
-	</tr>
-	<tr class="row2">
-		<td class="col0">2μl </td><td class="col1"> DNA</td>
-	</tr>
-	<tr class="row3">
-		<td class="col0">1μl </td><td class="col1"> EcoRV</td>
-	</tr>
-	<tr class="row4">
-		<td class="col0">10,5μl </td><td class="col1"> H2O</td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_42'> </div>
-<p>
-Digest at 37°C for 1h.
-Gel: undigested 203_2, 203_2, 203_1, 101_2, 101_1, 104_2, 104_1, ladder<br/>
-Img: 300-303
-</p>
-</div>
-<div class='secedit editbutton_section editbutton_41'> </div>
-<h3 class="sectionedit43" id="ligation_of_sgrnas_13_into_cas9_gfp">Ligation of sgRNAs 1;3 into CAS9 GFP</h3>
-<div class="level3">
-<p>
-No colonies from 24.05.17<br/>
-Transformation with heatshock protocol of 10μl of remaining ligation from 24.05.17<br/>
-</p>
-</div>
-<div class='secedit editbutton_section editbutton_43'> </div>
-<h2 class="sectionedit44" id="section260517">26.05.17</h2>
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_44'> </div>
-<h3 class="sectionedit45" id="sitedirected_mutagenesis_sdm_q5_pcr_with_sgrna_overhang_primers">Sitedirected mutagenesis (SDM) Q5 PCR with sgRNA overhang primers.</h3>
-<div class="level3">
-<p>
-Test with sgRNA 1;20<br/>
-</p>
-<div class="table sectionedit46"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">Volume</th><th class="col1">Component</th><td class="col2"></td>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0">12.5μl</td><td class="col1"> Q5 2x Mastermix</td><td class="col2"></td>
-	</tr>
-	<tr class="row2">
-		<td class="col0">1.25μl</td><td class="col1"> 10μM Fwd Primer</td><td class="col2"></td>
-	</tr>
-	<tr class="row3">
-		<td class="col0">1.25μl</td><td class="col1"> 10μM Rev Primer</td><td class="col2"></td>
-	</tr>
-	<tr class="row4">
-		<td class="col0">1μl </td><td class="col1">40 ng/μl CAS9-GFP</td><td class="col2"></td>
 	</tr>
 	<tr class="row5">
-		<td class="col0">9μl </td><td class="col1">H2O</td><td class="col2"></td>
+		<td class="col0">2000</td><td class="col1">100</td><td class="col2">28.8</td><td class="col3">0.8</td><td class="col4">5.91</td><td class="col5">5.65</td><td class="col6">5.75</td><td class="col7">5.77</td><td class="col8">0.08</td>
 	</tr>
 	<tr class="row6">
-		<th class="col0">Temp.[°C]</th><th class="col1">Time</th><th class="col2">Cycles</th>
+		<td class="col0">2000</td><td class="col1">120</td><td class="col2">34.2</td><td class="col3">0.9</td><td class="col4">4.99</td><td class="col5">4.96</td><td class="col6">4.93</td><td class="col7">4.96</td><td class="col8">0.02</td>
 	</tr>
 	<tr class="row7">
-		<td class="col0">98</td><td class="col1">30 sec.</td><td class="col2"></td>
+		<td class="col0">2000</td><td class="col1">140</td><td class="col2">40</td><td class="col3">1</td><td class="col4">4.41</td><td class="col5">4.37</td><td class="col6">4.36</td><td class="col7">4.38</td><td class="col8">0.02</td>
 	</tr>
 	<tr class="row8">
-		<td class="col0">98</td><td class="col1">10 sec.</td><td class="col2"></td>
+		<td class="col0">2000</td><td class="col1">160</td><td class="col2">45</td><td class="col3">1</td><td class="col4">4.31</td><td class="col5">4.12</td><td class="col6">4.10</td><td class="col7">4.18</td><td class="col8">0.07</td>
 	</tr>
 	<tr class="row9">
-		<td class="col0">54/61</td><td class="col1">45 sec.</td><td class="col2"></td>
+		<td class="col0">2000</td><td class="col1">180</td><td class="col2">50</td><td class="col3">1</td><td class="col4">3.98</td><td class="col5">3.95</td><td class="col6">3.98</td><td class="col7">3.97</td><td class="col8">0.01</td>
-	</tr>
-	<tr class="row10">
-		<td class="col0">72</td><td class="col1">4.5 min.</td><td class="col2"></td>
-	</tr>
-	<tr class="row11">
-		<td class="col0">98</td><td class="col1">10 sec.</td><td class="col2">24</td>
-	</tr>
-	<tr class="row12">
-		<td class="col0">64/70</td><td class="col1">30 sec.</td><td class="col2">24</td>
-	</tr>
-	<tr class="row13">
-		<td class="col0">72</td><td class="col1">3 min.</td><td class="col2">24</td>
-	</tr>
-	<tr class="row14">
-		<td class="col0">4</td><td class="col1"> 2 min.</td><td class="col2"></td>
 	</tr>
 </table></div>
-<div class='secedit editbutton_table editbutton_46'> </div><ul>
+<div class='secedit editbutton_table editbutton_4'> </div>
-<li class="level1"><div class="li"> Agarose Gel showed bands at wrong size (4kb)<br/>
-</div>
-</li>
-<li class="level1"><div class="li"> Gelextraction</div>
-</li>
-<li class="level1"><div class="li"> not further used</div>
-</li>
-</ul>
-</div>
-<div class='secedit editbutton_section editbutton_45'> </div>
-<h3 class="sectionedit47" id="ligation_of_sgrna_in_cas9-gfp">Ligation of sgRNA in CAS9-GFP</h3>
-<div class="level3">
-<ul>
-<li class="level1"><div class="li"> Few colonies observed</div>
-</li>
-<li class="level1"><div class="li"> Plates thrown out because nonsense</div>
-</li>
-</ul>
-</div>
-<div class='secedit editbutton_section editbutton_47'> </div>
-<h2 class="sectionedit48" id="section270517">27.05.17</h2>
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_48'> </div>
-<h3 class="sectionedit49" id="test_of_midi_prep_kit">Test of Midi Prep Kit</h3>
-<div class="level3">
 <p>
-use of different Kits &amp; performed at 2 Labs<br/>
+<br/>
-similar concentrations observed<br/>
+<em class="u"><strong>Resulting Titration curve for lactic acid in RPMI 1640:</strong></em><br/>
 </p>
+<div class="table sectionedit5"><table class="inline">
-</div>
-<div class='secedit editbutton_section editbutton_49'> </div>
-<h2 class="sectionedit50" id="section280517">28.05.17</h2>
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_50'> </div>
-<h3 class="sectionedit51" id="sdm_pcr_sgrna1_cas9gfp_gradient">SDM PCR sgRNA1 CAS9GFP gradient</h3>
-<div class="level3">
-<div class="table sectionedit52"><table class="inline">
 	<thead>
 	<tr class="row0">
-		<th class="col0">Volume</th><th class="col1">Component</th>
+		<th class="col0">Titration curve with pH vs. c(lactic acid)</th><th class="col1">Titration curve with pH vs. V(lactic acid 0.6 M) per ml</th>
 	</tr>
 	</thead>
 	<tr class="row1">
-		<td class="col0">12.5μl</td><td class="col1"> Q5 2x Mastermix</td>
+		<td class="col0"><img style="max-height: 400px" src="https://static.igem.org/mediawiki/2017/e/ee/T-Freiburg-6_6_2017_titration.jpeg"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=titration_final.jpg" class="media" title="titration_final.jpg"><img src="/igem2017/lib/exe/fetch.php?w=400&amp;tok=b008f9&amp;media=titration_final.jpg" class="mediacenter" alt="" width="400" /></a></td><td class="col1"><img style="max-height: 400px" src="https://static.igem.org/mediawiki/2017/1/18/T-Freiburg-6_6_2017_titration2.jpeg"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=titration_final_volume.jpg" class="media" title="titration_final_volume.jpg"><img src="/igem2017/lib/exe/fetch.php?w=400&amp;tok=ab0a8c&amp;media=titration_final_volume.jpg" class="mediacenter" alt="" width="400" /></a></td>
-	</tr>
-	<tr class="row2">
-		<td class="col0">1.25μl</td><td class="col1"> 10μM Fwd Primer</td>
-	</tr>
-	<tr class="row3">
-		<td class="col0">1.25μl</td><td class="col1"> 10μM Rev Primer</td>
-	</tr>
-	<tr class="row4">
-		<td class="col0">1μl </td><td class="col1">40 ng/μl CAS9-GFP</td>
-	</tr>
-	<tr class="row5">
-		<td class="col0">9μl </td><td class="col1">H2O</td>
 	</tr>
 </table></div>
-<div class='secedit editbutton_table editbutton_52'> </div>
+<div class='secedit editbutton_table editbutton_5'> </div>
-<p>
-Program, gradient PCR:<br/>
-</p>
-<div class="table sectionedit53"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">Temp.[°C]</th><th class="col1">Time</th><th class="col2">Cycles</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0">98</td><td class="col1">30 sec.</td><td class="col2"></td>
-	</tr>
-	<tr class="row2">
-		<td class="col0">98</td><td class="col1">10 sec.</td><td class="col2"></td>
-	</tr>
-	<tr class="row3">
-		<td class="col0">61/62/63/64</td><td class="col1">45 sec.</td><td class="col2"></td>
-	</tr>
-	<tr class="row4">
-		<td class="col0">72</td><td class="col1">4.5 min.</td><td class="col2"></td>
-	</tr>
-	<tr class="row5">
-		<td class="col0">98</td><td class="col1">10 sec.</td><td class="col2">24</td>
-	</tr>
-	<tr class="row6">
-		<td class="col0">70/71/72/73</td><td class="col1">6.5 min.</td><td class="col2">24</td>
-	</tr>
-	<tr class="row7">
-		<td class="col0">4</td><td class="col1"> 2 min.</td><td class="col2"></td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_53'> </div><ul>
-<li class="level1"><div class="li"> Agarose gel: loading low temp → high temp</div>
-</li>
-<li class="level1"><div class="li"> 8kb band observed (expected 9kb)</div>
-</li>
-</ul>
 </div>
-<div class='secedit editbutton_section editbutton_51'> </div>
+<div class='secedit editbutton_section editbutton_3'> </div>
-<h2 class="sectionedit54" id="section290517">29.05.17</h2>
+<h2 class="sectionedit6" id="section130617">13.06.17</h2>
 <div class="level2">
 </div>
-<div class='secedit editbutton_section editbutton_54'> </div>
+<div class='secedit editbutton_section editbutton_6'> </div>
-<h3 class="sectionedit55" id="sdm_pcr_for_all_sgrnas_cas9-gfp">SDM PCR for all sgRNAs CAS9-GFP</h3>
+<h3 class="sectionedit7" id="cell_survival_and_cfp-stability_at_different_ph-values">Cell survival and GFP-stability at different pH-values</h3>
-<div class="level3">
-<div class="table sectionedit56"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">Volume</th><th class="col1">Component</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0">12.5μl</td><td class="col1"> Q5 2x Mastermix</td>
-	</tr>
-	<tr class="row2">
-		<td class="col0">1.25μl</td><td class="col1"> 10μM Fwd Primer</td>
-	</tr>
-	<tr class="row3">
-		<td class="col0">1.25μl</td><td class="col1"> 10μM Rev Primer</td>
-	</tr>
-	<tr class="row4">
-		<td class="col0">1μl </td><td class="col1">40 ng/μl CAS9-GFP</td>
-	</tr>
-	<tr class="row5">
-		<td class="col0">9μl </td><td class="col1">H2O</td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_56'> </div>
-<p>
-<strong>program: sgRNAs 1;5;20</strong>
-</p>
-<div class="table sectionedit57"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">Temp.[°C]</th><th class="col1">Time</th><th class="col2">Cycles</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0">98</td><td class="col1">30 sec.</td><td class="col2"></td>
-	</tr>
-	<tr class="row2">
-		<td class="col0">98</td><td class="col1">10 sec.</td><td class="col2"></td>
-	</tr>
-	<tr class="row3">
-		<td class="col0">63</td><td class="col1">45 sec.</td><td class="col2"></td>
-	</tr>
-	<tr class="row4">
-		<td class="col0">72</td><td class="col1">4.5 min.</td><td class="col2"></td>
-	</tr>
-	<tr class="row5">
-		<td class="col0">98</td><td class="col1">10 sec.</td><td class="col2">24</td>
-	</tr>
-	<tr class="row6">
-		<td class="col0">72</td><td class="col1">6.5 min.</td><td class="col2">24</td>
-	</tr>
-	<tr class="row7">
-		<td class="col0">4</td><td class="col1"> 2 min.</td><td class="col2"></td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_57'> </div>
-<p>
-<strong>program: sgRNAs 3;4;9</strong>
-</p>
-<div class="table sectionedit58"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">Temp.[°C]</th><th class="col1">Time</th><th class="col2">Cycles</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0">98</td><td class="col1">30 sec.</td><td class="col2"></td>
-	</tr>
-	<tr class="row2">
-		<td class="col0">98</td><td class="col1">10 sec.</td><td class="col2"></td>
-	</tr>
-	<tr class="row3">
-		<td class="col0">59</td><td class="col1">45 sec.</td><td class="col2"></td>
-	</tr>
-	<tr class="row4">
-		<td class="col0">72</td><td class="col1">4.5 min.</td><td class="col2"></td>
-	</tr>
-	<tr class="row5">
-		<td class="col0">98</td><td class="col1">10 sec.</td><td class="col2">24</td>
-	</tr>
-	<tr class="row6">
-		<td class="col0">72</td><td class="col1">8 min.</td><td class="col2">24</td>
-	</tr>
-	<tr class="row7">
-		<td class="col0">4</td><td class="col1"> 2 min.</td><td class="col2"></td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_58'> </div>
-</div>
-<div class='secedit editbutton_section editbutton_55'> </div>
-<h2 class="sectionedit59" id="section300517">30.05.17</h2>
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_59'> </div>
-<h3 class="sectionedit60" id="bsmbi_digest_of_lenti-cas9-puro_ag_cathomen">BsmBI digest of lenti-CAS9-puro (AG Cathomen)</h3>
 <div class="level3">
 <p>
-μl ~80ng/μl lenti-CAS9<br/>
+used cells: Jurkat with CMV_GFP<br/>
-μl  10x NEB 3.1 Buffer<br/>
+cell density: 1 mio cells/ml<br/>
-.2μl BsmBI<br/>
+incubation times: 3 h, 6 h, 12 h<br/>
-μl  H2O<br/>
 </p>
 <p>
-@55°C; 1h incubation.<br/>
+<em class="u"><strong>Execution:</strong></em><br/>
 </p>
+<ol>
-</div>
+<li class="level1"><div class="li"> count cells</div>
-<div class='secedit editbutton_section editbutton_60'> </div>
-<h3 class="sectionedit61" id="sdm_pcr_of_sgrna1_vs_ctrl">SDM PCR of sgRNA1 vs. ctrl</h3>
-<div class="level3">
-<p>
-Image: 315
-</p>
-</div>
-<div class='secedit editbutton_section editbutton_61'> </div>
-<h2 class="sectionedit62" id="section310517">31.05.17</h2>
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_62'> </div>
-<h3 class="sectionedit63" id="sdm_pcr_for_sgrna520">SDM PCR for sgRNA5;20</h3>
-<div class="level3">
-<div class="table sectionedit64"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">Volume</th><th class="col1">Component</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0">12.5μl</td><td class="col1"> Q5 2x Mastermix</td>
-	</tr>
-	<tr class="row2">
-		<td class="col0">1.25μl</td><td class="col1"> 10μM Fwd Primer</td>
-	</tr>
-	<tr class="row3">
-		<td class="col0">1.25μl</td><td class="col1"> 10μM Rev Primer</td>
-	</tr>
-	<tr class="row4">
-		<td class="col0">1μl </td><td class="col1">40 ng/μl CAS9-GFP</td>
-	</tr>
-	<tr class="row5">
-		<td class="col0">9μl </td><td class="col1">H2O</td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_64'> </div>
-<p>
-<strong>program: sgRNAs 5;20</strong>
-</p>
-<div class="table sectionedit65"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">Temp.[°C]</th><th class="col1">Time</th><th class="col2">Cycles</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0">98</td><td class="col1">30 sec.</td><td class="col2"></td>
-	</tr>
-	<tr class="row2">
-		<td class="col0">98</td><td class="col1">10 sec.</td><td class="col2"></td>
-	</tr>
-	<tr class="row3">
-		<td class="col0">59</td><td class="col1">45 sec.</td><td class="col2"></td>
-	</tr>
-	<tr class="row4">
-		<td class="col0">72</td><td class="col1">4.5 min.</td><td class="col2"></td>
-	</tr>
-	<tr class="row5">
-		<td class="col0">98</td><td class="col1">10 sec.</td><td class="col2">24</td>
-	</tr>
-	<tr class="row6">
-		<td class="col0">72</td><td class="col1">8 min.</td><td class="col2">24</td>
-	</tr>
-	<tr class="row7">
-		<td class="col0">4</td><td class="col1"> 2 min.</td><td class="col2"></td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_65'> </div><div class="table sectionedit66"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0" colspan="2">Mix 1: sgRNA 1;5;20</th><th class="col2" colspan="2">Mix 2: sgRNA 1;5;20</th><th class="col4" colspan="2">Mix 3: sgRNA1</th>
-	</tr>
-	<tr class="row1">
-		<th class="col0">volume</th><th class="col1">component</th><th class="col2">volume</th><th class="col3">component</th><th class="col4">volume</th><th class="col5">component</th>
-	</tr>
-	</thead>
-	<tr class="row2">
-		<td class="col0">12.5μl</td><td class="col1"> Q5 2x Mastermix</td><td class="col2">5μl</td><td class="col3">Q5 Buffer</td><td class="col4">12.5μl</td><td class="col5"> Q5 2x Mastermix</td>
-	</tr>
-	<tr class="row3">
-		<td class="col0">1.25μl</td><td class="col1"> 10μM Fwd Primer</td><td class="col2">0.125μl</td><td class="col3">40mM dNTPs</td><td class="col4">1.25μl</td><td class="col5"> 10μM Fwd Primer</td>
-	</tr>
-	<tr class="row4">
-		<td class="col0">1.25μl</td><td class="col1"> 10μM Rev Primer</td><td class="col2">1.25μl</td><td class="col3"> 10μM Fwd Primer</td><td class="col4">1.25μl</td><td class="col5"> 10μM Rev Primer</td>
-	</tr>
-	<tr class="row5">
-		<td class="col0">1μl </td><td class="col1">40 ng/μl CAS9-GFP</td><td class="col2">1μl</td><td class="col3">400ng/μl CAS9-GFP</td><td class="col4">1μl </td><td class="col5">40 ng/μl CAS9-GFP</td>
-	</tr>
-	<tr class="row6">
-		<td class="col0"> </td><td class="col1"> </td><td class="col2">0.25μl</td><td class="col3">Q5 Polymerase</td><td class="col4"> </td><td class="col5"> </td>
-	</tr>
-	<tr class="row7">
-		<td class="col0">9μl </td><td class="col1">H2O</td><td class="col2">16,125μl</td><td class="col3">H2O</td><td class="col4">9μl </td><td class="col5">H2O</td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_66'> </div>
-</div>
-<div class='secedit editbutton_section editbutton_63'> </div>
-<h3 class="sectionedit67" id="gelextraction_of_sgrna1_in_cas9-gfp">Gelextraction of sgRNA1 in CAS9-GFP</h3>
-<div class="level3">
-</div>
-<div class='secedit editbutton_section editbutton_67'> </div>
-<h3 class="sectionedit68" id="bsmbi_digest_of_lenti-cas9-puro">BsmBI digest of lenti-CAS9-puro</h3>
-<div class="level3">
-<div class="table sectionedit69"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0">20μl</td><td class="col1">~70ng/μl plasmid</td>
-	</tr>
-	<tr class="row2">
-		<td class="col0">2.5μl</td><td class="col1">10x NEB 3.1</td>
-	</tr>
-	<tr class="row3">
-		<td class="col0">0.2μl</td><td class="col1">BsmBI</td>
-	</tr>
-	<tr class="row4">
-		<td class="col0">12.3μl</td><td class="col1">H2O</td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_69'> </div>
-</div>
-<div class='secedit editbutton_section editbutton_68'> </div>
-              </div>
-              <button class="accordion">June</button>
-        <div id="vanish" class="panel panel-default">
-<h2 class="sectionedit70" id="section010617">01.06.17</h2>
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_70'> </div>
-<h3 class="sectionedit71" id="cas9-gfp-sgrna1_t4_ligation_dpni_digest">CAS9-GFP-sgRNA1 T4 ligation &amp; DpnI digest</h3>
-<div class="level3">
-<div class="table sectionedit72"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0">4μl</td><td class="col1"> Gel extract</td>
-	</tr>
-	<tr class="row2">
-		<td class="col0">1μl</td><td class="col1"> T4 ligase</td>
-	</tr>
-	<tr class="row3">
-		<td class="col0">2μl</td><td class="col1"> 10x T4 Buffer</td>
-	</tr>
-	<tr class="row4">
-		<td class="col0">13μl</td><td class="col1">H2O</td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_72'> </div><div class="table sectionedit73"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0">2μl</td><td class="col1"> Ligation</td>
-	</tr>
-	<tr class="row2">
-		<td class="col0">1μl</td><td class="col1"> Cutsmart 10x Buffer</td>
-	</tr>
-	<tr class="row3">
-		<td class="col0">1μl</td><td class="col1"> DpnI</td>
-	</tr>
-	<tr class="row4">
-		<td class="col0">6μl</td><td class="col1"> H2O</td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_73'> </div>
-<p>
-Transformation of XL10gold with heatshock protocol<br/>
-</p>
-<ul>
-<li class="level1"><div class="li"> Ligated digest of CAS9-GFP-sgRNA1</div>
 </li>
-<li class="level1"><div class="li"> pipette tip of CAS9-GFP stock</div>
+<li class="level1"><div class="li"> centrifuge cells</div>
 </li>
-</ul>
+<li class="level1"><div class="li"> resuspend cell pellet in RPMI 1640 (target-concentration: 2 mio cells/ml)</div>
-</div>
-<div class='secedit editbutton_section editbutton_71'> </div>
-<h3 class="sectionedit74" id="maxi_prep_kit_of_ko-kit_plasmids">Maxi Prep Kit of KO-KIT plasmids</h3>
-<div class="level3">
-<p>
-Zymo Maxi Prep Kit Zentrifugationsprotocol<br/>
-ml LB-Amp overnight culture<br/>
-</p>
-<ul>
-<li class="level1"><div class="li"> scramble plasmid: 144.3 ng/μl (400μl)<br/>
-</div>
 </li>
-<li class="level1"><div class="li"> plasmid 203: 134.9 ng/μl (400μl)<br/>
+<li class="level1"><div class="li"> mix 1.5 ml RPMI 1640 + calculated volume of lactic acid (0.6 M) + 500 µl cell suspension on 12-well-plate (Table 1)</div>
-</div>
 </li>
-<li class="level1"><div class="li"> plasmid 104: 115.0 ng/μl (400μl)<br/>
+<li class="level1"><div class="li"> incubate for defined time in the CO<sub>2</sub>-incubator</div>
-</div>
 </li>
-</ul>
+<li class="level1"><div class="li"> centrifuge samples</div>
+</li>
-</div>
+<li class="level1"><div class="li"> resuspend cell pellets in PBS FACS buffer (500 µl)</div>
-<div class='secedit editbutton_section editbutton_74'> </div>
-<h2 class="sectionedit75" id="section020617">02.06.17</h2>
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_75'> </div>
-<h3 class="sectionedit76" id="smd_pcr_cas9-gfp_sgrna_1520ctrl">SMD PCR CAS9-GFP sgRNA 1;5;20;ctrl</h3>
-<div class="level3">
-<p>
-protocol as previously; control with primers for sgRNA20<br/>
-<img src="https://static.igem.org/mediawiki/2017/e/e1/T%E2%80%93FREIBURG%E2%80%93322.png" width="500px">
-</p>
-</div>
-<div class='secedit editbutton_section editbutton_76'> </div>
-<h3 class="sectionedit77" id="bsmbi_digest_of_lenti-cas9-puro1">BsmBI digest of lenti-CAS9-puro</h3>
-<div class="level3">
-<div class="table sectionedit78"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0">20μl</td><td class="col1">~70ng/μl plasmid</td>
-	</tr>
-	<tr class="row2">
-		<td class="col0">2.5μl</td><td class="col1">10x NEB 3.1</td>
-	</tr>
-	<tr class="row3">
-		<td class="col0">0.2μl</td><td class="col1">BsmBI</td>
-	</tr>
-	<tr class="row4">
-		<td class="col0">12.3μl</td><td class="col1">H2O</td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_78'> </div>
-<p>
-samples taken at 2; 2.5; 3 hours<br/>
-<img src="https://static.igem.org/mediawiki/2017/c/cd/T%E2%80%93FREIBURG%E2%80%93324.png" width="500px">
-</p>
-</div>
-<div class='secedit editbutton_section editbutton_77'> </div>
-<h3 class="sectionedit79" id="sdm_pcr_cas9-gfp-sgrna5">SDM PCR CAS9-GFP-sgRNA5</h3>
-<div class="level3">
-<p>
-labbook weird O.o<br/>
-</p>
-</div>
-<div class='secedit editbutton_section editbutton_79'> </div>
-<h3 class="sectionedit80" id="inoculation_of_5ml_culture_lb-amp_with_xl10gold">Inoculation of 5ml culture LB-Amp with XL10gold</h3>
-<div class="level3">
-<ul>
-<li class="level1"><div class="li"> 5 colonies sgRNA1</div>
 </li>
-<li class="level1"><div class="li"> 2 CAS9-GFP empty</div>
+<li class="level1"><div class="li"> FACS analysis</div>
 </li>
-</ul>
+<li class="level1"><div class="li"> used medium was stored at 4°C for pH-test next day</div>
+</li>
-</div>
+</ol>
-<div class='secedit editbutton_section editbutton_80'> </div>
+<div class="table sectionedit8"><table class="inline">
-<h2 class="sectionedit81" id="section030617">03.06.17</h2>
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_81'> </div>
-<h3 class="sectionedit82" id="bsmbi_digest_of_lenti-cas9-puro2">BsmBI digest of lenti-CAS9-puro</h3>
-<div class="level3">
-<div class="table sectionedit83"><table class="inline">
 	<thead>
 	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
+		<th class="col0">pH</th><th class="col1">V(lactic acid, 0.6 M) [µl]</th><th class="col2">RPMI 1640 [µl]</th>
 	</tr>
 	</thead>
 	<tr class="row1">
-		<td class="col0">20μl</td><td class="col1">~70ng/μl plasmid</td>
+		<td class="col0">5.0</td><td class="col1">120</td><td class="col2">2000</td>
 	</tr>
 	<tr class="row2">
-		<td class="col0">2.5μl</td><td class="col1">10x NEB 3.1</td>
+		<td class="col0">5.5</td><td class="col1">107</td><td class="col2">2000</td>
 	</tr>
 	<tr class="row3">
-		<td class="col0">0.5μl</td><td class="col1">BsmBI</td>
+		<td class="col0">6.0</td><td class="col1">92</td><td class="col2">2000</td>
 	</tr>
 	<tr class="row4">
-		<td class="col0">7μl</td><td class="col1">H2O</td>
+		<td class="col0">6.5</td><td class="col1">68</td><td class="col2">2000</td>
+	</tr>
+	<tr class="row5">
+		<td class="col0">7.0</td><td class="col1">0</td><td class="col2">2000</td>
 	</tr>
 </table></div>
-<div class='secedit editbutton_table editbutton_83'> </div>
+<div class='secedit editbutton_table editbutton_8'> </div>
 <p>
-digest @ 55°C for 2h<br/>
+<br/>
-μl [50 ng/μl] plasmid loaded as control<br/>
+<em class="u"><strong>Results:</strong></em><br/>
-Image: 333/334<br/>
 </p>
-</div>
-<div class='secedit editbutton_section editbutton_82'> </div>
-<h3 class="sectionedit84" id="midi_prep">Midi Prep</h3>
-<div class="level3">
-<ul>
-<li class="level1"><div class="li"> CAS9-GFP empty</div>
-</li>
-<li class="level1"><div class="li"> CAS9-GFP sgRNA1</div>
-</li>
-</ul>
 <p>
-Production of glycerol stocks 1x CAS9-GFP; 5x CAS9-GFP-sgRNA1<br/>
+→ Jurkat cells can survive in medium with pH ≥ 6.0<br/>
 </p>
-</div>
-<div class='secedit editbutton_section editbutton_84'> </div>
-<h3 class="sectionedit85" id="sdm_pcr_of_cas9-gfp_sgrna5">SDM PCR of CAS9-GFP sgRNA5</h3>
-<div class="level3">
 <p>
-Protocol same as 26.05.17<br/>
+<strong>cell survival</strong>
-Dilution series of plasmid stock: 200ng/μl; 100ng/μl; 50ng/μl; 25ng/μl; 12.5ng/μl; 6.25ng/μl<br/>
-New 1:10 dilution of primer stock was made<br/>
 </p>
+<div class="table sectionedit9"><table class="inline">
-</div>
-<div class='secedit editbutton_section editbutton_85'> </div>
-<h2 class="sectionedit86" id="section040617">04.06.17</h2>
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_86'> </div>
-<h3 class="sectionedit87" id="pcr_egfp-fragment_for_sgen_plasmid">PCR eGFP-fragment for SGEN plasmid</h3>
-<div class="level3">
-<div class="table sectionedit88"><table class="inline">
 	<thead>
 	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
+		<th class="col0">after 3h</th><th class="col1">after 6h</th><th class="col2">after 12h</th>
 	</tr>
 	</thead>
 	<tr class="row1">
-		<td class="col0">10μl</td><td class="col1">5x Q5 Buffer</td>
+		<td class="col0"><img style="max-height: 150px" src="https://static.igem.org/mediawiki/2017/8/85/T-Freiburg-6_13_2017_cell_survival_CFP_stability_1.jpeg"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=photo_2017-06-14_15-15-20.jpg" class="media" title="photo_2017-06-14_15-15-20.jpg"><img src="/igem2017/lib/exe/fetch.php?w=300&amp;tok=78e39b&amp;media=photo_2017-06-14_15-15-20.jpg" class="mediacenter" alt="" width="300" /></a></td><td class="col1"><img style="max-height: 150px" src="https://static.igem.org/mediawiki/2017/a/a4/T-Freiburg-6_13_2017_cell_survival_CFP_stability_2.jpeg"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=photo_2017-06-14_15-15-36.jpg" class="media" title="photo_2017-06-14_15-15-36.jpg"><img src="/igem2017/lib/exe/fetch.php?w=300&amp;tok=9b5f60&amp;media=photo_2017-06-14_15-15-36.jpg" class="mediacenter" alt="" width="300" /></a></td><td class="col2"><img style="max-height: 150px" src="https://static.igem.org/mediawiki/2017/c/ca/T-Freiburg-6_13_2017_cell_survival_CFP_stability_3.jpeg"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=photo_2017-06-14_15-15-42.jpg" class="media" title="photo_2017-06-14_15-15-42.jpg"><img src="/igem2017/lib/exe/fetch.php?w=300&amp;tok=291bdb&amp;media=photo_2017-06-14_15-15-42.jpg" class="mediacenter" alt="" width="300" /></a></td>
-	</tr>
-	<tr class="row2">
-		<td class="col0">1μl</td><td class="col1">10mM dNTPs</td>
-	</tr>
-	<tr class="row3">
-		<td class="col0">2.5μl</td><td class="col1">10mM FW primer (olG17_111)</td>
-	</tr>
-	<tr class="row4">
-		<td class="col0">2.5μl</td><td class="col1">10mM RW primer (olG17_110)</td>
-	</tr>
-	<tr class="row5">
-		<td class="col0">0.2μl</td><td class="col1">Template (plG_9)</td>
-	</tr>
-	<tr class="row6">
-		<td class="col0">0.5μl</td><td class="col1">Q5 Polymerase</td>
-	</tr>
-	<tr class="row7">
-		<td class="col0">33.3μl</td><td class="col1"> H2O</td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_88'> </div><div class="table sectionedit89"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">Temp. [°C]</th><th class="col1">Time</th><th class="col2">Cycles</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0">98</td><td class="col1" colspan="2">30 sec.</td>
-	</tr>
-	<tr class="row2">
-		<td class="col0">98</td><td class="col1">10 sec.</td><td class="col2">33</td>
-	</tr>
-	<tr class="row3">
-		<td class="col0">67</td><td class="col1">30 sec.</td><td class="col2">33</td>
-	</tr>
-	<tr class="row4">
-		<td class="col0">72</td><td class="col1">20 sec.</td><td class="col2">33</td>
-	</tr>
-	<tr class="row5">
-		<td class="col0">72</td><td class="col1" colspan="2">2 min.</td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_89'> </div>
-</div>
-<div class='secedit editbutton_section editbutton_87'> </div>
-<h3 class="sectionedit90" id="pcr_sden_short_fragment_for_sgen_plasmid">PCR SDEN short fragment for SGEN plasmid</h3>
-<div class="level3">
-<div class="table sectionedit91"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0">10μl</td><td class="col1">5x Q5 Buffer</td>
-	</tr>
-	<tr class="row2">
-		<td class="col0">1μl</td><td class="col1">10mM dNTPs</td>
-	</tr>
-	<tr class="row3">
-		<td class="col0">2.5μl</td><td class="col1">10mM FW primer (olG17_109)</td>
-	</tr>
-	<tr class="row4">
-		<td class="col0">2.5μl</td><td class="col1">10mM RW primer (olG17_108)</td>
-	</tr>
-	<tr class="row5">
-		<td class="col0">0.2μl</td><td class="col1">Template (1754 base fragment of SGEN)</td>
-	</tr>
-	<tr class="row6">
-		<td class="col0">0.5μl</td><td class="col1">Q5 Polymerase</td>
-	</tr>
-	<tr class="row7">
-		<td class="col0">33.3μl</td><td class="col1"> H2O</td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_91'> </div><div class="table sectionedit92"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">Temp. [°C]</th><th class="col1">Time</th><th class="col2">Cycles</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0">98</td><td class="col1" colspan="2">30 sec.</td>
-	</tr>
-	<tr class="row2">
-		<td class="col0">98</td><td class="col1">10 sec.</td><td class="col2">33</td>
-	</tr>
-	<tr class="row3">
-		<td class="col0">67</td><td class="col1">30 sec.</td><td class="col2">33</td>
-	</tr>
-	<tr class="row4">
-		<td class="col0">72</td><td class="col1">20 sec.</td><td class="col2">33</td>
-	</tr>
-	<tr class="row5">
-		<td class="col0">72</td><td class="col1" colspan="2">2 min.</td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_92'> </div>
-</div>
-<div class='secedit editbutton_section editbutton_90'> </div>
-<h3 class="sectionedit93" id="sden_bamhi_digest">SDEN BamHI digest</h3>
-<div class="level3">
-<div class="table sectionedit94"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0">2μl</td><td class="col1">Plasmid (SDEN)</td>
-	</tr>
-	<tr class="row2">
-		<td class="col0">3μl</td><td class="col1">Cut Smart 10x Buffer</td>
-	</tr>
-	<tr class="row3">
-		<td class="col0">1μl</td><td class="col1">BamHI</td>
-	</tr>
-	<tr class="row4">
-		<td class="col0">24μl</td><td class="col1">H2O</td>
 	</tr>
 </table></div>
-<div class='secedit editbutton_table editbutton_94'> </div>
+<div class='secedit editbutton_table editbutton_9'> </div>
 <p>
-Digest at 37°C for 1 hour. Expected fragment lenght: 4768 bp, 2386 bp.<br/>
+<a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=cell_survival.jpg" class="media" title="cell_survival.jpg"><img src="/igem2017/lib/exe/fetch.php?w=400&amp;tok=01ecfc&amp;media=cell_survival.jpg" class="mediacenter" alt="" width="400" /></a>
-Gel extraction of GFP-fragment. Obtained concentration: 7,8 ng/μl.<br/>
 </p>
-</div>
+<img style="max-height: 400px" src="https://static.igem.org/mediawiki/2017/0/0c/T-Freiburg_6_13_2017_cell_survival_GFP_stability_4.jpeg">
-<div class='secedit editbutton_section editbutton_93'> </div>
-<h3 class="sectionedit95" id="cas9-gfp-sgrna_5_ligation">CAS9-GFP-sgRNA 5 Ligation</h3>
-<div class="level3">
-<div class="table sectionedit96"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0" colspan="2">Mix 1: 200ng/μl template (12kb fragment)</th><th class="col2" colspan="2">Mix 2: 200ng/μl template (10kb fragment)</th><th class="col4" colspan="2">Mix 3: 50ng/μl template (10kb fragment)</th>
-	</tr>
-	<tr class="row1">
-		<th class="col0">volume</th><th class="col1">component</th><th class="col2">volume</th><th class="col3">component</th><th class="col4">volume</th><th class="col5">component</th>
-	</tr>
-	</thead>
-	<tr class="row2">
-		<td class="col0">8μl</td><td class="col1"> purified PCR product</td><td class="col2">8μl</td><td class="col3"> purified PCR product</td><td class="col4">8μl</td><td class="col5"> purified PCR product</td>
-	</tr>
-	<tr class="row3">
-		<td class="col0">2μl</td><td class="col1"> T4 ligase 10x buffer</td><td class="col2">2μl</td><td class="col3"> T4 ligase 10x buffer</td><td class="col4">2μl</td><td class="col5"> T4 ligase 10x buffer</td>
-	</tr>
-	<tr class="row4">
-		<td class="col0">1μl</td><td class="col1"> T4 ligase</td><td class="col2">1μl</td><td class="col3"> T4 ligase</td><td class="col4">1μl</td><td class="col5"> T4 ligase</td>
-	</tr>
-	<tr class="row5">
-		<td class="col0">9μl </td><td class="col1">H2O</td><td class="col2">9μl </td><td class="col3">H2O</td><td class="col4">9μl </td><td class="col5">H2O</td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_96'> </div>
-<p>
-Plasmids were ligated for 2 hours at 16°C.
-</p>
 <p>
-Ligated plasmid was digested with DpnI for 1 hour at 37°C.<br/>
+<strong>GFP fluorescence</strong>
 </p>
-<div class="table sectionedit97"><table class="inline">
+<div class="table sectionedit10"><table class="inline">
 	<thead>
 	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
+		<th class="col0">after 3h</th><th class="col1">after 6h</th><th class="col2">after 12h</th>
 	</tr>
 	</thead>
 	<tr class="row1">
-		<td class="col0">4μl</td><td class="col1"> Ligated plasmid</td>
+		<td class="col0"><img style="max-height: 150px" src="https://static.igem.org/mediawiki/2017/4/47/T-Freiburg-6_13_2017_cell_survival_CFP_stability_5.jpeg"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=photo_2017-06-14_15-15-48.jpg" class="media" title="photo_2017-06-14_15-15-48.jpg"><img src="/igem2017/lib/exe/fetch.php?w=300&amp;tok=9c7865&amp;media=photo_2017-06-14_15-15-48.jpg" class="mediacenter" alt="" width="300" /></a></td><td class="col1"><img style="max-height: 200px" src="https://static.igem.org/mediawiki/2017/8/86/T-Freiburg-6_13_2017_cell_survival_CFP_stability_6.jpeg"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=photo_2017-06-14_15-15-56.jpg" class="media" title="photo_2017-06-14_15-15-56.jpg"><img src="/igem2017/lib/exe/fetch.php?w=300&amp;tok=17b2bf&amp;media=photo_2017-06-14_15-15-56.jpg" class="mediacenter" alt="" width="300" /></a></td><td class="col2"><img style="max-height: 200px" src="https://static.igem.org/mediawiki/2017/5/5f/T-Freiburg-6_13_2017_cell_survival_CFP_stability_7.jpeg"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=photo_2017-06-14_15-16-01.jpg" class="media" title="photo_2017-06-14_15-16-01.jpg"><img src="/igem2017/lib/exe/fetch.php?w=300&amp;tok=1a7641&amp;media=photo_2017-06-14_15-16-01.jpg" class="mediacenter" alt="" width="300" /></a></td>
-	</tr>
-	<tr class="row2">
-		<td class="col0">1μl</td><td class="col1"> Cut Smart 10x buffer</td>
-	</tr>
-	<tr class="row3">
-		<td class="col0">1μl</td><td class="col1"> DpnI</td>
-	</tr>
-	<tr class="row4">
-		<td class="col0">4μl</td><td class="col1"> H2O</td>
 	</tr>
 </table></div>
-<div class='secedit editbutton_table editbutton_97'> </div>
+<div class='secedit editbutton_table editbutton_10'> </div>
-</div>
-<div class='secedit editbutton_section editbutton_95'> </div>
-<h3 class="sectionedit98" id="oligocloning_sgrna1_3_in_lenti-cas9-puromycin">Oligocloning sgRNA1/3 in lenti-CAS9-puromycin</h3>
-<div class="level3">
-<div class="table sectionedit99"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0">7μl</td><td class="col1"> 11kb fragment of BsmBI digested lenti-CAS9-puromycin plasmid</td>
-	</tr>
-	<tr class="row2">
-		<td class="col0">1μl</td><td class="col1">50nM dilution of annealed dsOligo (sgRNA1/3)</td>
-	</tr>
-	<tr class="row3">
-		<td class="col0">1μl</td><td class="col1"> T4 ligase 10x Buffer</td>
-	</tr>
-	<tr class="row4">
-		<td class="col0">1μl</td><td class="col1"> T4 ligase</td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_99'> </div>
 <p>
-As a control 1μl H2O was used instead of dsOligos.<br/>
+<a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=fluorescence_intensity.jpg" class="media" title="fluorescence_intensity.jpg"><img src="/igem2017/lib/exe/fetch.php?w=400&amp;tok=c9ebab&amp;media=fluorescence_intensity.jpg" class="mediacenter" alt="" width="400" /></a>
 </p>
-</div>
+<img style="max-height: 400px" src="https://static.igem.org/mediawiki/2017/d/d9/T%E2%80%93FREIBURG-6_13_2017_cell_survival_CFP_stability_8.jpeg">
-<div class='secedit editbutton_section editbutton_98'> </div>
-<h3 class="sectionedit100" id="transformation_of_xl10gold_via_heatshock_transformation">Transformation of XL10gold via heatshock transformation</h3>
-<div class="level3">
-<ul>
-<li class="level1"><div class="li"> 3 Ligated &amp; digested CAS9-GFP-sgRNA5 plasmid</div>
-</li>
-<li class="level1"><div class="li"> sgRNA1/3/control of lenti-CAS9-puromycin plasmid</div>
-</li>
-</ul>
-<p>
-Heatshock transformation was performed accoarding to the heatshock protocol.
-</p>
 </div>
-<div class='secedit editbutton_section editbutton_100'> </div>
+<div class='secedit editbutton_section editbutton_7'> </div>
-<h2 class="sectionedit101" id="section050617">05.06.17</h2>
+<h2 class="sectionedit11" id="section140617">14.06.17</h2>
 <div class="level2">
 </div>
-<div class='secedit editbutton_section editbutton_101'> </div>
+<div class='secedit editbutton_section editbutton_11'> </div>
-<h3 class="sectionedit102" id="digest_of_lenti-cas9-puromycin">Digest of lenti-CAS9-puromycin</h3>
+<h3 class="sectionedit12" id="ph-measurement_of_used_medium_130617">pH-measurement of used medium (13.06.17)</h3>
 <div class="level3">
-<div class="table sectionedit103"><table class="inline">
+<div class="table sectionedit13"><table class="inline">
 	<thead>
 	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
+		<th class="col0">set pH</th><th class="col1">6 h</th><th class="col2">12 h</th>
 	</tr>
 	</thead>
 	<tr class="row1">
-		<td class="col0">15μl</td><td class="col1"> Plasmid [c=70 ng/μl]</td>
+		<td class="col0">pH = 5.0</td><td class="col1">4.67</td><td class="col2">4.78</td>
 	</tr>
 	<tr class="row2">
-		<td class="col0">0,5μl</td><td class="col1"> BsmBI</td>
+		<td class="col0">pH = 5.5</td><td class="col1">5.19</td><td class="col2">5.43</td>
 	</tr>
 	<tr class="row3">
-		<td class="col0">2,5μl</td><td class="col1"> NEB 3.1 10x buffer</td>
+		<td class="col0">pH = 6.0</td><td class="col1">6.26</td><td class="col2">6.33</td>
 	</tr>
 	<tr class="row4">
-		<td class="col0">7μl</td><td class="col1"> H2O</td>
+		<td class="col0">pH = 6.5</td><td class="col1">6.95</td><td class="col2">6.76</td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_103'> </div>
-<p>
-Digest for 2 hours at 55°C.<br/>
-Gel: with EtBr.<br/>
-</p>
-</div>
-<div class='secedit editbutton_section editbutton_102'> </div>
-<h3 class="sectionedit104" id="sdm_pcr_for_cas9-gfp-shrna349">SDM PCR for CAS9-GFP-shRNA3;4;9</h3>
-<div class="level3">
-<div class="table sectionedit105"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0">12,5μl</td><td class="col1">Q5 MasterMix 2x</td>
-	</tr>
-	<tr class="row2">
-		<td class="col0">1,25μl</td><td class="col1">FW primer (for sgRNA3,4,9)</td>
-	</tr>
-	<tr class="row3">
-		<td class="col0">1,25μl</td><td class="col1">RW primer (for sgRNA3,4,9)</td>
-	</tr>
-	<tr class="row4">
-		<td class="col0">1μl</td><td class="col1">CAS9-GFP plasmid</td>
 	</tr>
 	<tr class="row5">
-		<td class="col0">9μl</td><td class="col1">H2O</td>
+		<td class="col0">untreated (pH = 7.25)</td><td class="col1">7.73</td><td class="col2">7.47</td>
 	</tr>
 </table></div>
-<div class='secedit editbutton_table editbutton_105'> </div>
+<div class='secedit editbutton_table editbutton_13'> </div>
 <p>
-PCR program: sgRNA insertion 2.<br/>
+<strong>Result:</strong>
+pH in incubation medium is rising over the time! Expected was a lowering, as the cells secrete lactic acid.
-</p>
-<div class="table sectionedit106"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">step</th><th class="col1">temperature (°C)</th><th class="col2">time</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0"> 1 </td><td class="col1"> 98.0 </td><td class="col2"> 00:30 </td>
-	</tr>
-	<tr class="row2">
-		<td class="col0"> 2 </td><td class="col1"> 98.0 </td><td class="col2"> 00:10 </td>
-	</tr>
-	<tr class="row3">
-		<td class="col0"> 3 </td><td class="col1"> 57.8 </td><td class="col2"> 00:45 </td>
-	</tr>
-	<tr class="row4">
-		<td class="col0"> 4 </td><td class="col1"> 70.8 </td><td class="col2"> 06:30 </td>
-	</tr>
-	<tr class="row5">
-		<td class="col0"> 5 </td><td class="col1"> 98.0 </td><td class="col2"> 00:10 </td>
-	</tr>
-	<tr class="row6">
-		<td class="col0"> 6 </td><td class="col1"> 70.8 </td><td class="col2"> 07:00 </td>
-	</tr>
-	<tr class="row7">
-		<td class="col0"> 7 </td><td class="col1"> 72.0 </td><td class="col2"> 10:00 </td>
-	</tr>
-	<tr class="row8">
-		<td class="col0"> 8 </td><td class="col1"> 72.0 </td><td class="col2"> pause </td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_106'> </div>
-<p>
-Steps 2 to 4 were repeated 1x and steps 5 to 6 were repeated 24x
 </p>
 </div>
-<div class='secedit editbutton_section editbutton_104'> </div>
+<div class='secedit editbutton_section editbutton_12'> </div>
-<h2 class="sectionedit107" id="section060617">06.06.17</h2>
+<h2 class="sectionedit14" id="section170617">17.06.17</h2>
 <div class="level2">
 </div>
-<div class='secedit editbutton_section editbutton_107'> </div>
+<div class='secedit editbutton_section editbutton_14'> </div>
-<h3 class="sectionedit108" id="miniprep_of_overnight_lenti-cas9-sgrna3">Miniprep of overnight lenti-CAS9-sgRNA3</h3>
+<h3 class="sectionedit15" id="investigation_of_the_ph-changes">Investigation of the pH-changes</h3>
 <div class="level3">
 <p>
-Miniprep was performed accoarding to the manufactors protocol.<br/>
+test of pH-change of RPMI 1640 at different conditions<br/>
 </p>
-</div>
-<div class='secedit editbutton_section editbutton_108'> </div>
-<h3 class="sectionedit109" id="sdm_pcr_for_sgrna3_4_9">SDM PCR for sgRNA3,4,9</h3>
-<div class="level3">
-<div class="table sectionedit110"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0">1μl</td><td class="col1">CAS9-GFP plasmid</td>
-	</tr>
-	<tr class="row2">
-		<td class="col0">0,5μl</td><td class="col1">FW primer (for sgRNA3,4,9)</td>
-	</tr>
-	<tr class="row3">
-		<td class="col0">0,5μl</td><td class="col1">RW primer (olG_118)</td>
-	</tr>
-	<tr class="row4">
-		<td class="col0">2,5μl</td><td class="col1">10x Taq buffer</td>
-	</tr>
-	<tr class="row5">
-		<td class="col0">0,5μl</td><td class="col1">dNTPs</td>
-	</tr>
-	<tr class="row6">
-		<td class="col0">1μl</td><td class="col1">Taq (peqlab)</td>
-	</tr>
-	<tr class="row7">
-		<td class="col0">19,875μl</td><td class="col1">H2O</td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_110'> </div>
 <p>
-Annealing at 54°C, Elongation 72°C for 40sec., 30 cycles.<br/>
+tested conditions:
-</p>
-</div>
-<div class='secedit editbutton_section editbutton_109'> </div>
-<h2 class="sectionedit111" id="section070617">07.06.17</h2>
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_111'> </div>
-<h3 class="sectionedit112" id="gelelectrophoresis_of_pcrs">Gelelectrophoresis of PCRs</h3>
-<div class="level3">
-<p>
-SDM all failed.<br/>
-Colony PCR positive for
 </p>
 <ul>
-<li class="level1"><div class="li"> sgRNA5 in CAS9-GFP</div>
+<li class="level1"><div class="li"> incubation in stock RPMI 1640 or RPMI 1640 with lactic acid (pH = 6.0)</div>
 </li>
-<li class="level1"><div class="li"> sgRNA in lenti-CAS9-puromycin</div>
+<li class="level1"><div class="li"> medium with/without Jurkat cells (500k/ml)</div>
+</li>
+<li class="level1"><div class="li"> incubation at 37°C in incubator with CO<sub>2</sub> (5%) atmosphere or incubated with cap</div>
+</li>
+<li class="level1"><div class="li"> incubation at atmospheric conditions</div>
+</li>
+<li class="level1"><div class="li"> incubation time: 6 h</div>
 </li>
 </ul>
+<ol>
+<li class="level1"><div class="li"> make aliquots of RPMI 1640 (15 ml, pH = 6.0) and untreated RPMI 1640 (15 ml)</div>
+</li>
+<li class="level1"><div class="li"> aliquot 1: measure pH before incubation</div>
+</li>
+<li class="level1"><div class="li"> other aliquots: incubate at conditions described above<br/>
 </div>
-<div class='secedit editbutton_section editbutton_112'> </div>
+</li>
-<h3 class="sectionedit113" id="sdm_pcr_for_cas9-gfp-shrna3_4_9">SDM PCR for CAS9-GFP-shRNA3,4,9</h3>
+</ol>
-<div class="level3">
-<div class="table sectionedit114"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0">12,5μl</td><td class="col1">Q5 MasterMix 2x</td>
-	</tr>
-	<tr class="row2">
-		<td class="col0">1,25μl</td><td class="col1">FW primer (for sgRNA3,4,9)</td>
-	</tr>
-	<tr class="row3">
-		<td class="col0">1,25μl</td><td class="col1">RW primer (for sgRNA3,4,9)</td>
-	</tr>
-	<tr class="row4">
-		<td class="col0">1μl</td><td class="col1">CAS9-GFP plasmid</td>
-	</tr>
-	<tr class="row5">
-		<td class="col0">9μl</td><td class="col1">H2O</td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_114'> </div>
-<p>
-PCR program: sgRNA insertion 2.<br/>
-</p>
 <p>
-Gel: 363,364<br/>
+<strong>Results</strong>
-Loading: sgRNA9, sgRNA4, sgRNA3<br/>
 </p>
+<div class="table sectionedit16"><table class="inline">
-</div>
-<div class='secedit editbutton_section editbutton_113'> </div>
-<h2 class="sectionedit115" id="section080617">08.06.17</h2>
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_115'> </div>
-<h3 class="sectionedit116" id="colonie_pcr_for_sgrna_1_3_5">Colonie PCR for sgRNA 1,3,5</h3>
-<div class="level3">
-<div class="table sectionedit117"><table class="inline">
 	<thead>
 	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
+		<th class="col0">6 h, 37 °C, 5% CO<sub>2</sub></th><th class="col1">RPMI 1640 (pH = 6.0)</th><th class="col2">stock RPMI 1640</th>
 	</tr>
 	</thead>
 	<tr class="row1">
-		<td class="col0">1μl</td><td class="col1">Purified plasmid</td>
+		<td class="col0">pH before incubation</td><td class="col1">5.87</td><td class="col2">7.45</td>
 	</tr>
 	<tr class="row2">
-		<td class="col0">0,5μl</td><td class="col1">FW primer (for sgRNA1,3,5)</td>
+		<td class="col0">pH after incubation</td><td class="col1">6.46</td><td class="col2">7.68</td>
-	</tr>
-	<tr class="row3">
-		<td class="col0">0,5μl</td><td class="col1">RW primer (olG_118)</td>
-	</tr>
-	<tr class="row4">
-		<td class="col0">2,5μl</td><td class="col1">10x Taq buffer</td>
-	</tr>
-	<tr class="row5">
-		<td class="col0">0,5μl</td><td class="col1">dNTPs</td>
-	</tr>
-	<tr class="row6">
-		<td class="col0">0,125μl</td><td class="col1">Taq (peqlab)</td>
-	</tr>
-	<tr class="row7">
-		<td class="col0">19,875μl</td><td class="col1">H2O</td>
 	</tr>
 </table></div>
-<div class='secedit editbutton_table editbutton_117'> </div>
+<div class='secedit editbutton_table editbutton_16'> </div>
 <p>
-Picture: 373<br/>
+<br/>
 </p>
+<div class="table sectionedit17"><table class="inline">
-</div>
-<div class='secedit editbutton_section editbutton_116'> </div>
-<h2 class="sectionedit118" id="section090617">09.06.17</h2>
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_118'> </div>
-<h3 class="sectionedit119" id="digest_of_lenti-cas9-puromycin_with_bsmbi">Digest of lenti-CAS9-puromycin with BsmBI</h3>
-<div class="level3">
-<div class="table sectionedit120"><table class="inline">
 	<thead>
 	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
+		<th class="col0">6 h, 37 °C, 5% CO<sub>2</sub>, Jurkat cells</th><th class="col1">RPMI 1640 (pH = 6.0)</th><th class="col2">stock RPMI 1640</th>
 	</tr>
 	</thead>
 	<tr class="row1">
-		<td class="col0">15μl</td><td class="col1">lenti-CAS9-puromycin [c=70ng/μl]</td>
+		<td class="col0">pH before incubation</td><td class="col1">5.87</td><td class="col2">7.45</td>
 	</tr>
 	<tr class="row2">
-		<td class="col0">0,5μl</td><td class="col1">BsmBI</td>
+		<td class="col0">pH after incubation</td><td class="col1">6.48</td><td class="col2">7.70</td>
-	</tr>
-	<tr class="row3">
-		<td class="col0">2,5μl</td><td class="col1">NEB  3.1 10x Buffer</td>
-	</tr>
-	<tr class="row4">
-		<td class="col0">7μl</td><td class="col1"> H2O</td>
 	</tr>
 </table></div>
-<div class='secedit editbutton_table editbutton_120'> </div>
+<div class='secedit editbutton_table editbutton_17'> </div>
 <p>
-Digest for 2 hours at 55°C.<br/>
+<br/>
 </p>
+<div class="table sectionedit18"><table class="inline">
-</div>
-<div class='secedit editbutton_section editbutton_119'> </div>
-<h2 class="sectionedit121" id="section100617">10.06.17</h2>
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_121'> </div>
-<h3 class="sectionedit122" id="sdm_pcr_for_cas9-gfp-sgrna3_4_9">SDM PCR for CAS9-GFP-sgRNA3,4,9</h3>
-<div class="level3">
-<div class="table sectionedit123"><table class="inline">
 	<thead>
 	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
+		<th class="col0">6 h, 37 °C, with cap</th><th class="col1">RPMI 1640 (pH = 6.0)</th><th class="col2">stock RPMI 1640</th>
 	</tr>
 	</thead>
 	<tr class="row1">
-		<td class="col0">12,5μl</td><td class="col1">Q5 MasterMix 2x</td>
+		<td class="col0">pH before incubation</td><td class="col1">5.87</td><td class="col2">7.45</td>
 	</tr>
 	<tr class="row2">
-		<td class="col0">1,25μl</td><td class="col1">FW primer (for sgRNA3,4,9)</td>
+		<td class="col0">pH after incubation</td><td class="col1">6.63</td><td class="col2">8.46</td>
-	</tr>
-	<tr class="row3">
-		<td class="col0">1,25μl</td><td class="col1">RW primer (for sgRNA3,4,9)</td>
-	</tr>
-	<tr class="row4">
-		<td class="col0">1μl</td><td class="col1">CAS9-GFP plasmid</td>
-	</tr>
-	<tr class="row5">
-		<td class="col0">9μl</td><td class="col1">H2O</td>
 	</tr>
 </table></div>
-<div class='secedit editbutton_table editbutton_123'> </div>
+<div class='secedit editbutton_table editbutton_18'> </div>
 <p>
-PCR program: sgRNA insertion 2.<br/>
+<br/>
 </p>
+<div class="table sectionedit19"><table class="inline">
-</div>
-<div class='secedit editbutton_section editbutton_122'> </div>
-<h3 class="sectionedit124" id="ligation_of_sgrna_4_5_9_20_into_lenti-cas9-puromycin_plasmid">Ligation of sgRNA 4,5,9,20 into lenti-CAS9-puromycin plasmid</h3>
-<div class="level3">
-<div class="table sectionedit125"><table class="inline">
 	<thead>
 	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
+		<th class="col0">6 h, 25 °C, atmospheric conditions</th><th class="col1">RPMI 1640 (pH = 6.0)</th><th class="col2">stock RPMI 1640</th>
 	</tr>
 	</thead>
 	<tr class="row1">
-		<td class="col0">7μl</td><td class="col1"> 11kb fragment of BsmBI digested lenti-CAS9-puromycin plasmid</td>
+		<td class="col0">pH before incubation</td><td class="col1">5.87</td><td class="col2">7.45</td>
 	</tr>
 	<tr class="row2">
-		<td class="col0">1μl</td><td class="col1">50nM dilution of annealed dsOligo (sgRNA4/5/9/20)</td>
+		<td class="col0">pH after incubation</td><td class="col1">6.24</td><td class="col2">7.79</td>
-	</tr>
-	<tr class="row3">
-		<td class="col0">1μl</td><td class="col1"> T4 ligase 10x Buffer</td>
-	</tr>
-	<tr class="row4">
-		<td class="col0">1μl</td><td class="col1"> T4 ligase</td>
 	</tr>
 </table></div>
-<div class='secedit editbutton_table editbutton_125'> </div>
+<div class='secedit editbutton_table editbutton_19'> </div>
-<p>
-As a control 1μl H2O was used instead of dsOligos.<br/>
-</p>
 </div>
-<div class='secedit editbutton_section editbutton_124'> </div>
+<div class='secedit editbutton_section editbutton_15'> </div>
-<h3 class="sectionedit126" id="heatshock_transformation_of_xl10gold">Heatshock transformation of XL10gold</h3>
+<h2 class="sectionedit20" id="section190617">19.06.17</h2>
-<div class="level3">
+<div class="level2">
-<p>
-Heatshock transformation was performed accoarding to the heatshock protocol.<br/>
-</p>
 </div>
-<div class='secedit editbutton_section editbutton_126'> </div>
+<div class='secedit editbutton_section editbutton_20'> </div>
-<h3 class="sectionedit127" id="digest_of_lenti-cas9-puromycin_with_bsmbi1">Digest of lenti-CAS9-puromycin with BsmBI</h3>
+<h3 class="sectionedit21" id="ph_change_vs_incubation_time">pH change vs. incubation time</h3>
 <div class="level3">
-<div class="table sectionedit128"><table class="inline">
+<ol>
+<li class="level1"><div class="li"> set pH to 6.3</div>
+</li>
+<li class="level1"><div class="li"> prepare 2 ml aliquots of RPMI 1640 in falcon tubes</div>
+</li>
+<li class="level1"><div class="li"> incubate in CO<sub>2</sub> incubator for t (min) = 0, 10, 20, 30, 50</div>
+</li>
+<li class="level1"><div class="li"> measure pH directly after taking sample out of incubator</div>
+</li>
+<li class="level1"><div class="li"> measure pH one minute later</div>
+</li>
+</ol>
+<div class="table sectionedit22"><table class="inline">
 	<thead>
 	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
+		<th class="col0">time /min</th><th class="col1">pH direct</th><th class="col2">pH after 1 min</th>
 	</tr>
 	</thead>
 	<tr class="row1">
-		<td class="col0">15μl</td><td class="col1">lenti-CAS9-puromycin [c=70ng/μl]</td>
+		<td class="col0">0</td><td class="col1">6.32</td><td class="col2">6.32</td>
 	</tr>
 	<tr class="row2">
-		<td class="col0">0,5μl</td><td class="col1">BsmBI</td>
+		<td class="col0">10</td><td class="col1">6.03</td><td class="col2">6.25</td>
 	</tr>
 	<tr class="row3">
-		<td class="col0">2,5μl</td><td class="col1">NEB  3.1 10x Buffer</td>
+		<td class="col0">20</td><td class="col1">6.06</td><td class="col2">6.28</td>
 	</tr>
 	<tr class="row4">
-		<td class="col0">7μl</td><td class="col1"> H2O</td>
+		<td class="col0">30</td><td class="col1">5.88</td><td class="col2">6.36</td>
+	</tr>
+	<tr class="row5">
+		<td class="col0">50</td><td class="col1">6.36</td><td class="col2">6.65</td>
 	</tr>
 </table></div>
-<div class='secedit editbutton_table editbutton_128'> </div>
+<div class='secedit editbutton_table editbutton_22'> </div>
 <p>
-Digest for 2 hours at 55°C.<br/>
+pH is strongly rising after taking the sample out of the incubator. This may be caused by the solved CO<sub>2</sub>, that lowers the pH in the incubator. After taking the sample out of the incubator, the CO<sub>2</sub> goes to the gas phase which causes a rising of the pH. Additionally, the bicarbonate buffer is destabilized as the addition of lactic acid moves the equilibrium of the buffer to CO<sub>2</sub> + H<sub>2</sub>O. That leads to a higher pH than before the incubation.
 </p>
 </div>
-<div class='secedit editbutton_section editbutton_127'> </div>
-<h3 class="sectionedit129" id="agarose_gel_for_cas9-gfp-sgrna3_4_9">Agarose gel for CAS9-GFP-sgRNA3,4,9</h3>
-<div class="level3">
-<p>
-Pic:
-</p>
 </div>
-<div class='secedit editbutton_section editbutton_129'> </div>
+              <button class="accordion">July</button>
-<h2 class="sectionedit130" id="section110617">11.06.17</h2>
+         <div class="panel panel-default">
+<div class='secedit editbutton_section editbutton_21'> </div>
+<h2 class="sectionedit23" id="section040717">04.07.17</h2>
 <div class="level2">
 </div>
-<div class='secedit editbutton_section editbutton_130'> </div>
+<div class='secedit editbutton_section editbutton_23'> </div>
-<h3 class="sectionedit131" id="minipreps_of_cas9-gfp-sgrna1_cas9-gfp_lenti-cas9-puromycin">Minipreps of CAS9-GFP-sgRNA1, CAS9-GFP, lenti-CAS9-puromycin</h3>
+<h3 class="sectionedit24" id="vegf_dilution">VEGF dilution</h3>
 <div class="level3">
 <p>
-Minipreps were performed accoarding to the manufacturer&#039;s protocol.<br/>
+m<sub>0</sub> = 10 µg
+start concentration: solid state
+target concentration: 1 µg/ml
 </p>
+<ol>
+<li class="level1"><div class="li"> resuspend pellet in 100 µl ddH<sub>2</sub>O (stock solution, 100 µg/ml)</div>
+</li>
+<li class="level1"><div class="li"> dilute 1 µl of stock solution with 99 µl ddH<sub>2</sub>O (aliquots, 1 µg/ml)</div>
+</li>
+<li class="level1"><div class="li"> store aliquots at -20 °C</div>
+</li>
+</ol>
 </div>
-<div class='secedit editbutton_section editbutton_131'> </div>
+<div class='secedit editbutton_section editbutton_24'> </div>
-<h3 class="sectionedit132" id="sdm_pcr_for_cas9-gfp-sgrna3_4_91">SDM PCR for CAS9-GFP-sgRNA3,4,9</h3>
+<h2 class="sectionedit25" id="section060717">06.07.17</h2>
-<div class="level3">
-<div class="table sectionedit133"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0">12,5μl</td><td class="col1">Q5 MasterMix 2x</td>
-	</tr>
-	<tr class="row2">
-		<td class="col0">1,25μl</td><td class="col1">FW primer (for sgRNA3,4,9)</td>
-	</tr>
-	<tr class="row3">
-		<td class="col0">1,25μl</td><td class="col1">RW primer (for sgRNA3,4,9)</td>
-	</tr>
-	<tr class="row4">
-		<td class="col0">1μl</td><td class="col1">CAS9-GFP plasmid</td>
-	</tr>
-	<tr class="row5">
-		<td class="col0">9μl</td><td class="col1">H2O</td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_133'> </div>
-<p>
-PCR program: sgRNA insertion 2.<br/>
-</p>
-</div>
-<div class='secedit editbutton_section editbutton_132'> </div>
-<h2 class="sectionedit134" id="section120617">12.06.17</h2>
 <div class="level2">
 </div>
-<div class='secedit editbutton_section editbutton_134'> </div>
+<div class='secedit editbutton_section editbutton_25'> </div>
-<h3 class="sectionedit135" id="minipreps_of_lenti-cas9-puro-sgrna4_5_9_20">Minipreps of lenti-CAS9-puro-sgRNA4,5,9,20</h3>
+<h3 class="sectionedit26" id="pctla4_vs_vegf">pCTLA4 vs. [VEGF]</h3>
 <div class="level3">
 <p>
-Minipreps were performed accoarding to the manufacturer&#039;s protocol.<br/>
+<strong>Aim:</strong> determination of suitable [VEGF] and incubation time<br/>
 </p>
-</div>
-<div class='secedit editbutton_section editbutton_135'> </div>
-<h3 class="sectionedit136" id="gelextraction_of_cas9-gfp-sgrna3_4_9">Gelextraction of CAS9-GFP-sgRNA3,4,9</h3>
-<div class="level3">
 <p>
-Gelextraction was performed accoarding to the manufacturer&#039;s protocol.<br/>
+<strong>tested cells</strong>: HEK with transient pCTLA4-GFP (pIG17_022 (330 bp); pIG17_023 (380 bp)),PEI transfected<br/>
-</p>
+[VEGF](ng/ml) = 0.0, 0.5, 5.0, 10.0<br/>
-</div>
+t (h) = 3, 8, 24<br/>
-<div class='secedit editbutton_section editbutton_136'> </div>
-<h2 class="sectionedit137" id="section130617">13.06.17</h2>
-<div class="level2">
-</div>
+controls: CMV_GFP (pIG_009), untransfected cells
-<div class='secedit editbutton_section editbutton_137'> </div>
+</p>
-<h3 class="sectionedit138" id="cas9-gfp-sgrna_5_ligation1">CAS9-GFP-sgRNA 5 Ligation</h3>
-<div class="level3">
-<div class="table sectionedit139"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0">8μl</td><td class="col1"> purified PCR product</td>
-	</tr>
-	<tr class="row2">
-		<td class="col0">2μl</td><td class="col1"> T4 ligase 10x buffer</td>
-	</tr>
-	<tr class="row3">
-		<td class="col0">1μl</td><td class="col1"> T4 ligase</td>
-	</tr>
-	<tr class="row4">
-		<td class="col0">9μl </td><td class="col1">H2O</td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_139'> </div>
-<p>
-Plasmids were ligated for 2 hours at room temperature.
-Ligated plasmid was digested with DpnI for 1 hour at 37°C.<br/>
-</p>
-<div class="table sectionedit140"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0">4μl</td><td class="col1"> Ligated plasmid</td>
-	</tr>
-	<tr class="row2">
-		<td class="col0">1μl</td><td class="col1"> Cut Smart 10x buffer</td>
-	</tr>
-	<tr class="row3">
-		<td class="col0">1μl</td><td class="col1"> DpnI</td>
-	</tr>
-	<tr class="row4">
-		<td class="col0">4μl</td><td class="col1"> H2O</td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_140'> </div>
 <p>
-μl ligated and digested plasmid was transformed using the heatshock protocol.<br/>
+Execution:
 </p>
+<ol>
-</div>
+<li class="level1"><div class="li"> cells were PEI transfected by cell culture on 05.07.17</div>
-<div class='secedit editbutton_section editbutton_138'> </div>
+</li>
-<h3 class="sectionedit141" id="heatshock_transformation_of_xl10gold_with_cas9-gfp-sgrna3_4_9">Heatshock transformation of XL10gold with CAS9-GFP-sgRNA3,4,9</h3>
+<li class="level1"><div class="li"> 24h after PEI transfection: replace old medium by RPMI 1640 (2 ml / well), add VEGF (1 µg/ml)</div>
-<div class="level3">
+</li>
+<li class="level1"><div class="li"> after incubation time: analysis via fluorescence microscope and FACS</div>
+</li>
+</ol>
 <p>
-Transformation of XL10gold was performed accoarding to the heatshock protocol.<br/>
+<strong><em class="u">Results</em></strong>:
+<a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=histogram_results.jpg" class="media" title="histogram_results.jpg"><img src="/igem2017/lib/exe/fetch.php?w=600&amp;tok=68fc80&amp;media=histogram_results.jpg" class="mediacenter" alt="" width="600" /></a>
 </p>
-</div>
+<img style="max-height: 1600px" src="https://static.igem.org/mediawiki/2017/7/76/T-Freiburg-7_6_2017_pCTLA4_vs_VEGF.jpeg">
-<div class='secedit editbutton_section editbutton_141'> </div>
-<h2 class="sectionedit142" id="section140617">14.06.17</h2>
-<div class="level2">
 </div>
-<div class='secedit editbutton_section editbutton_142'> </div>
+<div class='secedit editbutton_section editbutton_26'> </div>
-<h3 class="sectionedit143" id="sdm_pcr_for_cas9-gfp-sgrna20">SDM PCR for CAS9-GFP-sgRNA20</h3>
+<h2 class="sectionedit27" id="section180717">18.07.17</h2>
-<div class="level3">
-<div class="table sectionedit144"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0">12,5μl</td><td class="col1">Q5 MasterMix 2x</td>
-	</tr>
-	<tr class="row2">
-		<td class="col0">1,25μl</td><td class="col1">FW primer (for sgRNA20)</td>
-	</tr>
-	<tr class="row3">
-		<td class="col0">1,25μl</td><td class="col1">RW primer (olG_118)</td>
-	</tr>
-	<tr class="row4">
-		<td class="col0">1μl</td><td class="col1">CAS9-GFP plasmid</td>
-	</tr>
-	<tr class="row5">
-		<td class="col0">9μl</td><td class="col1">H2O</td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_144'> </div>
-<p>
-PCR program: sgRNA insertion 2.<br/>
-</p>
-</div>
-<div class='secedit editbutton_section editbutton_143'> </div>
-<h2 class="sectionedit145" id="section150617">15.06.17</h2>
 <div class="level2">
 </div>
-<div class='secedit editbutton_section editbutton_145'> </div>
+<div class='secedit editbutton_section editbutton_27'> </div>
-<h3 class="sectionedit146" id="gelextraction_of_cas9-gfp-sgrna20">Gelextraction of CAS9-GFP-sgRNA20</h3>
+<h3 class="sectionedit28" id="pei_transfection_of_plasmids_pig17_022_23_37_86">PEI transfection of plasmids pIG17_022/23/37/86</h3>
 <div class="level3">
 <p>
-Gelextraction was performed accoarding to the manufacturer&#039;s protocol.<br/>
+used cells: HEK cells<br/>
 </p>
+<div class="table sectionedit29"><table class="inline">
-</div>
-<div class='secedit editbutton_section editbutton_146'> </div>
-<h3 class="sectionedit147" id="gibson-assambly">Gibson-Assambly</h3>
-<div class="level3">
-<div class="table sectionedit148"><table class="inline">
 	<thead>
 	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
+		<th class="col0">Plasmid number</th><th class="col1">component</th>
 	</tr>
 	</thead>
 	<tr class="row1">
-		<td class="col0">1,8μl</td><td class="col1"> SDEN backbone</td>
+		<td class="col0">pIG17_022</td><td class="col1">Ctla4(380bp)-GFP</td>
 	</tr>
 	<tr class="row2">
-		<td class="col0">1,4μl</td><td class="col1"> SDEN fragment</td>
+		<td class="col0">pIG17_023</td><td class="col1">Ctla4(330bp)-GFP</td>
 	</tr>
 	<tr class="row3">
-		<td class="col0">1,8μl</td><td class="col1"> GFP</td>
+		<td class="col0">pIG17_037</td><td class="col1">Ctla4(330bp)-GFP-CMV-mCherry</td>
 	</tr>
 	<tr class="row4">
-		<td class="col0">5μl</td><td class="col1"> assembly-mix</td>
+		<td class="col0">pIG17_086</td><td class="col1">Ctla4(380bp)-GFP-CMV-mCherry</td>
 	</tr>
 </table></div>
-<div class='secedit editbutton_table editbutton_148'> </div><ul>
+<div class='secedit editbutton_table editbutton_29'> </div><ol>
-<li class="level1"><div class="li"> 15 min. at 55°C</div>
+<li class="level1"><div class="li"> Split the cells 1:5 into 10 cm plate (on 21.04.17)<br/>
+</div>
 </li>
-<li class="level1"><div class="li"> thaw Dh5α on ice</div>
+<li class="level1"><div class="li"> Thaw PEI reagent at RT<br/>
+</div>
 </li>
-<li class="level1"><div class="li"> add 5μl Gibson-mix</div>
+<li class="level1"><div class="li"> Dilute plasmid in 1 ml serum free DMEM (2.5 µg from each plasmid)<br/>
+</div>
 </li>
-<li class="level1"><div class="li"> incubate on ice for 10 min.</div>
+<li class="level1"><div class="li"> Add 15 µl PEI(1 µg/µl) to the mix while vortexing (PEI:DNA = 3:1)<br/>
+</div>
 </li>
-<li class="level1"><div class="li"> add 900μl LB-media</div>
+<li class="level1"><div class="li"> incubate 15 min at RT<br/>
+</div>
 </li>
-<li class="level1"><div class="li"> incubate for 1 hour at 37°C</div>
+<li class="level1"><div class="li"> Add the mix to the culture<br/>
+</div>
 </li>
-<li class="level1"><div class="li"> spread on LB-Amp plate</div>
+<li class="level1"><div class="li"> incubate for 24 h<br/>
+</div>
 </li>
-</ul>
+</ol>
 </div>
-<div class='secedit editbutton_section editbutton_147'> </div>
+<div class='secedit editbutton_section editbutton_28'> </div>
-<h2 class="sectionedit149" id="section160617">16.06.17</h2>
+<h2 class="sectionedit30" id="section190717">19.07.17</h2>
 <div class="level2">
 </div>
-<div class='secedit editbutton_section editbutton_149'> </div>
+<div class='secedit editbutton_section editbutton_30'> </div>
-<h3 class="sectionedit150" id="cas9-gfp-sgrna20_ligation">CAS9-GFP-sgRNA20 Ligation</h3>
+<h3 class="sectionedit31" id="vegf_test">VEGF test</h3>
 <div class="level3">
-<div class="table sectionedit151"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0">8μl</td><td class="col1"> purified PCR product</td>
-	</tr>
-	<tr class="row2">
-		<td class="col0">2μl</td><td class="col1"> T4 ligase 10x buffer</td>
-	</tr>
-	<tr class="row3">
-		<td class="col0">1μl</td><td class="col1"> T4 ligase</td>
-	</tr>
-	<tr class="row4">
-		<td class="col0">9μl </td><td class="col1">H2O</td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_151'> </div>
 <p>
-Plasmids were ligated for 2 hours at room temperature.
+used cells: PEI transfected cells from 19.07.17 with pIG17_022/023/037/086<br/>
 </p>
+<ol>
+<li class="level1"><div class="li"> add following VEGF concentrations to the cells: 0 ng/ml, 0.5 ng/ml, 5 ng/ml, 10 ng/ml, 50 ng/ml<br/>
 </div>
-<div class='secedit editbutton_section editbutton_150'> </div>
-<h3 class="sectionedit152" id="restriction_digest_of_sden-backbone_bamhi">Restriction digest of SDEN-Backbone BamHI</h3>
-<div class="level3">
-<div class="table sectionedit153"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0">5μl</td><td class="col1">SDEN</td>
-	</tr>
-	<tr class="row2">
-		<td class="col0">2μl</td><td class="col1">CutSmart 10x Buffer</td>
-	</tr>
-	<tr class="row3">
-		<td class="col0">1μl</td><td class="col1">BamHI</td>
-	</tr>
-	<tr class="row4">
-		<td class="col0">12μl</td><td class="col1">H2O</td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_153'> </div><ul>
-<li class="level1"><div class="li"> Incubate for 2 hours at 37°C</div>
 </li>
-<li class="level1"><div class="li"> Incubate for 20 min. at 65°C</div>
+<li class="level1"><div class="li"> incubate for 24 h<br/>
+</div>
 </li>
-<li class="level1"><div class="li"> Dephosphorylation:</div>
+</ol>
-</li>
-</ul>
+<p>
-<div class="table sectionedit154"><table class="inline">
+fluorescence microscope:<br/>
-	<thead>
-	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0">10μl</td><td class="col1"> Antarctic phosphotase buffer</td>
-	</tr>
-	<tr class="row2">
-		<td class="col0">1μl</td><td class="col1"> Antarctic Phosphotase</td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_154'> </div><ul>
-<li class="level1"><div class="li"> Incubation for 10 min. at 37°C</div>
-</li>
-<li class="level1"><div class="li"> Gel purification (1% agarose, 115V, 30 min.)</div>
-</li>
-</ul>
-</div>
+</p>
-<div class='secedit editbutton_section editbutton_152'> </div>
+<div class="table sectionedit32"><table class="inline">
-<h3 class="sectionedit155" id="gibson-assambly1">Gibson-Assambly</h3>
-<div class="level3">
-<div class="table sectionedit156"><table class="inline">
 	<thead>
 	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
+		<th class="col0">construct</th><th class="col1">bright field</th><th class="col2">GFP</th>
 	</tr>
 	</thead>
 	<tr class="row1">
-		<td class="col0">1,8μl</td><td class="col1"> SDEN backbone</td>
+		<td class="col0">positive control<br/>
+CMV-CFP</td><td class="col1"><img style="max-height: 300px" src="https://static.igem.org/mediawiki/2017/2/2c/T-Freiburg-7_19_2017_VEGF_test_1.png"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=gfp_pos_vis_black.png" class="media" title="gfp_pos_vis_black.png"><img src="/igem2017/lib/exe/fetch.php?w=400&amp;tok=64f251&amp;media=gfp_pos_vis_black.png" class="mediacenter" alt="" width="400" /></a></td><td class="col2"><img style="max-height: 300px" src="https://static.igem.org/mediawiki/2017/a/a2/T-Freiburg-7_19_2017_VEGF_test_2.png"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=gfp_pos_black.png" class="media" title="gfp_pos_black.png"><img src="/igem2017/lib/exe/fetch.php?w=400&amp;tok=70b3f6&amp;media=gfp_pos_black.png" class="mediacenter" alt="" width="400" /></a></td>
 	</tr>
 	<tr class="row2">
-		<td class="col0">1,4μl</td><td class="col1"> SDEN fragment</td>
+		<td class="col0">negative control</td><td class="col1"><img style="max-height: 300px" src="https://static.igem.org/mediawiki/2017/f/fb/T-Freiburg-7_19_2017_VEGF_test_3.png"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=neg_vis_black.png" class="media" title="neg_vis_black.png"><img src="/igem2017/lib/exe/fetch.php?w=400&amp;tok=f5f53c&amp;media=neg_vis_black.png" class="mediacenter" alt="" width="400" /></a></td><td class="col2"><img style="max-height: 300px" src="https://static.igem.org/mediawiki/2017/6/63/7_19_2017_VEGF_test_4.png"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=neg_black.png" class="media" title="neg_black.png"><img src="/igem2017/lib/exe/fetch.php?w=400&amp;tok=7a2af5&amp;media=neg_black.png" class="mediacenter" alt="" width="400" /></a></td>
 	</tr>
 	<tr class="row3">
-		<td class="col0">1,8μl</td><td class="col1"> GFP</td>
-	</tr>
-	<tr class="row4">
-		<td class="col0">5μl</td><td class="col1"> assembly-mix</td>
 	</tr>
 </table></div>
-<div class='secedit editbutton_table editbutton_156'> </div><ul>
+<div class='secedit editbutton_table editbutton_32'> </div>
-<li class="level1"><div class="li"> 15 min. at 55°C</div>
+</div>
-</li>
+<div class='secedit editbutton_section editbutton_31'> </div>
-<li class="level1"><div class="li"> thaw Dh5α on ice</div>
+</div>
-</li>
-<li class="level1"><div class="li"> add 5μl Gibson-mix</div>
-</li>
-<li class="level1"><div class="li"> incubate on ice for 10 min.</div>
-</li>
-<li class="level1"><div class="li"> add 900μl LB-media</div>
-</li>
-<li class="level1"><div class="li"> incubate for 1 hour at 37°C</div>
-</li>
-<li class="level1"><div class="li"> spread on LB-Amp plate</div>
-</li>
-</ul>
-</div>
+ <button class="accordion">August</button>
-<div class='secedit editbutton_section editbutton_155'> </div>
+         <div class="panel panel-default">
-<h2 class="sectionedit157" id="section170617">17.06.17</h2>
+<h2 class="sectionedit33" id="section050817">05.08.17</h2>
 <div class="level2">
 </div>
-<div class='secedit editbutton_section editbutton_157'> </div>
+<div class='secedit editbutton_section editbutton_33'> </div>
-<h3 class="sectionedit158" id="colony_pcr">Colony PCR</h3>
+<h3 class="sectionedit34" id="interlab_study_transformation">Interlab Study transformation</h3>
 <div class="level3">
-<ul>
-<li class="level1"><div class="li"> for plasmid CAS9-GFP-sgRNA3/9</div>
-</li>
-<li class="level1"><div class="li"> lenti-CAS9-puro-sgRNA1/3</div>
-</li>
-<li class="level1"><div class="li"> positive control RNA5_col3</div>
-</li>
-</ul>
 <p>
-Annealing changed to 58°C<br/>
+- heat shock transformation of DH5-α with plasmids from Kit plate 7<br/>
 </p>
+<div class="table sectionedit35"><table class="inline">
-</div>
-<div class='secedit editbutton_section editbutton_158'> </div>
-<h3 class="sectionedit159" id="digest_of_cas9-gfp-sgrna20_with_dpni">digest of CAS9-GFP-sgRNA20 with DpnI</h3>
-<div class="level3">
-<div class="table sectionedit160"><table class="inline">
 	<thead>
 	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
+		<th class="col0">well</th><th class="col1">approach</th>
 	</tr>
 	</thead>
 	<tr class="row1">
-		<td class="col0">4μl</td><td class="col1">Ligation</td>
+		<td class="col0">21B</td><td class="col1">positive control</td>
 	</tr>
 	<tr class="row2">
-		<td class="col0">1μl</td><td class="col1">Cut Smart 10x buffer</td>
+		<td class="col0">21D</td><td class="col1">negative control</td>
 	</tr>
 	<tr class="row3">
-		<td class="col0">1μl</td><td class="col1">DpnI</td>
+		<td class="col0">21F</td><td class="col1">Device 1</td>
 	</tr>
 	<tr class="row4">
-		<td class="col0">4μl</td><td class="col1">H2O</td>
+		<td class="col0">21H</td><td class="col1">Device 2</td>
 	</tr>
-</table></div>
+	<tr class="row5">
-<div class='secedit editbutton_table editbutton_160'> </div>
+		<td class="col0">21J</td><td class="col1">Device 3</td>
-<p>
-Digest for 1 hour at 27°C. 10μl were transformed into XL10gold using the heatshock protocol.<br/>
-</p>
-</div>
-<div class='secedit editbutton_section editbutton_159'> </div>
-<h3 class="sectionedit161" id="annealing_of_sgrna_oligos1">Annealing of sgRNA oligos</h3>
-<div class="level3">
-<div class="table sectionedit162"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
 	</tr>
-	</thead>
+	<tr class="row6">
-	<tr class="row1">
+		<td class="col0">21L</td><td class="col1">Device 4</td>
-		<td class="col0">5μl</td><td class="col1">100mM of each Fwd and Rev ssOligos</td>
 	</tr>
-	<tr class="row2">
+	<tr class="row7">
-		<td class="col0">10μl</td><td class="col1">10x NEB2.1 buffer</td>
+		<td class="col0">21N</td><td class="col1">Device 5</td>
 	</tr>
-	<tr class="row3">
+	<tr class="row8">
-		<td class="col0">80μl</td><td class="col1">H2O</td>
+		<td class="col0">21P</td><td class="col1">Device 6</td>
 	</tr>
 </table></div>
-<div class='secedit editbutton_table editbutton_162'> </div>
+<div class='secedit editbutton_table editbutton_35'> </div>
+</div>
+<div class='secedit editbutton_section editbutton_34'> </div>
+<h3 class="sectionedit36" id="interlab_study_od600_calibration_measurements">Interlab Study OD600 calibration measurements</h3>
+<div class="level3">
 <p>
-min. at 95°C cooling down in heatblock for 2h.<br/>
+- measure absorbance at 600 nm of H20 (100 µl) and LUDOX-S40 (100 µl)
+- each four replicates<br/>
 </p>
+<div class="table sectionedit37"><table class="inline">
-</div>
-<div class='secedit editbutton_section editbutton_161'> </div>
-<h3 class="sectionedit163" id="bsmbi_digest_of_lenti-cas9-puromycin">BsmBI digest of lenti-CAS9-puromycin</h3>
-<div class="level3">
-<div class="table sectionedit164"><table class="inline">
 	<thead>
 	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
+		<th class="col0">replicate</th><th class="col1">LUDOX-S40</th><th class="col2">H2O</th>
 	</tr>
 	</thead>
 	<tr class="row1">
-		<td class="col0">1μl</td><td class="col1">lenti-CAS9-puromycin</td>
+		<td class="col0">1</td><td class="col1">0,043</td><td class="col2 rightalign">	0,035</td>
 	</tr>
 	<tr class="row2">
-		<td class="col0">1,5μl</td><td class="col1">10x NEB 3.1 buffer</td>
+		<td class="col0">2</td><td class="col1">0,049</td><td class="col2 rightalign">	0,036</td>
 	</tr>
 	<tr class="row3">
-		<td class="col0">0,5μl</td><td class="col1">BsmBI</td>
+		<td class="col0">3</td><td class="col1">0,047</td><td class="col2 rightalign">	0,035</td>
 	</tr>
 	<tr class="row4">
-		<td class="col0">12μl</td><td class="col1">H2O</td>
+		<td class="col0">4</td><td class="col1">0,052</td><td class="col2 rightalign">	0,035</td>
 	</tr>
 </table></div>
-<div class='secedit editbutton_table editbutton_164'> </div>
+<div class='secedit editbutton_table editbutton_37'> </div>
-<p>
-Digest for 2 hours at 55°C.<br/>
-</p>
 </div>
-<div class='secedit editbutton_section editbutton_163'> </div>
+<div class='secedit editbutton_section editbutton_36'> </div>
-<h3 class="sectionedit165" id="miniprep_of_sgen-gibson_colony">Miniprep of SGEN-Gibson Colony</h3>
+<h3 class="sectionedit38" id="interlab_study_fluorescein_standard_curve_measurements">Interlab Study fluorescein standard curve measurements</h3>
 <div class="level3">
 <p>
-Minipreps were performed accoarding to the manufacturer&#039;s protocol.<br/>
+- centrifuge fluorescein<br/>
-Testdigest with NotI not succesful.<br/>
+- dilute fluorescein in PBS to a final concentration of 50 µM<br/>
-</p>
+- add PBS (100 µl) into well A2-A12, B2-B12, C2-C12, D2-D12 of a 96-well-plate<br/>
-</div>
+- add fluorescein stock solution (200 µl, 50µM) into well A1, B1, C1, D1<br/>
-<div class='secedit editbutton_section editbutton_165'> </div>
-<h2 class="sectionedit166" id="section180617">18.06.17</h2>
-<div class="level2">
-</div>
+- transfer 100 µl from well A1 into well A2, pipett up and down<br/>
-<div class='secedit editbutton_section editbutton_166'> </div>
-<h3 class="sectionedit167" id="ligation_of_sgrna_scramble_into_lenti-cas9-puromycin">Ligation of sgRNA scramble into lenti-CAS9-puromycin</h3>
-<div class="level3">
-<div class="table sectionedit168"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0">7μl</td><td class="col1"> 11kb fragment of BsmBI digested lenti-CAS9-puromycin plasmid</td>
-	</tr>
-	<tr class="row2">
-		<td class="col0">1μl</td><td class="col1">50nM dilution of annealed dsOligo (sgRNAscramble)</td>
-	</tr>
-	<tr class="row3">
-		<td class="col0">1μl</td><td class="col1"> T4 ligase 10x Buffer</td>
-	</tr>
-	<tr class="row4">
-		<td class="col0">1μl</td><td class="col1"> T4 ligase</td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_168'> </div>
-<p>
-μl were used for heatshock transformation.<br/>
-</p>
+- transfer 100 µl from well A2 into well A3, pipett up and down<br/>
-</div>
+- do the same till the end of the row<br/>
-<div class='secedit editbutton_section editbutton_167'> </div>
-<h3 class="sectionedit169" id="heatshock_transformation_of_xl10gold1">Heatshock transformation of XL10gold</h3>
-<div class="level3">
-<p>
+- transfer 100 µl from every last well into the liquid waste<br/>
-Heatshock transformation was performed accoarding to the heatshock protocol.<br/>
-</p>
+- repeat these steps for rpw B, C and D<br/>
-</div>
-<div class='secedit editbutton_section editbutton_169'> </div>
-<h2 class="sectionedit170" id="section190617">19.06.17</h2>
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_170'> </div>
-<h3 class="sectionedit171" id="miniprep_of_7_sgen-gibson_colonies">Miniprep of 7 SGEN-Gibson colonies</h3>
-<div class="level3">
-<p>
-Minipreps were performed accoarding to the manufacturer&#039;s protocol.<br/>
-Positive colonies were observed for colony 1,4,5 and 7.<br/>
+- measure the fluorescence intensity in the plate reader<br/>
 </p>
+<div class="table sectionedit39"><table class="inline">
-</div>
-<div class='secedit editbutton_section editbutton_171'> </div>
-<h3 class="sectionedit172" id="sdm_pcr_for_cas9-gfp-sgrnascramble">SDM PCR for CAS9-GFP-sgRNAscramble</h3>
-<div class="level3">
-<div class="table sectionedit173"><table class="inline">
 	<thead>
 	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
+		<th class="col0">uM Fluorescein</th><th class="col1">50,00</th><th class="col2">25</th><th class="col3">12,5</th><th class="col4">6,25</th><th class="col5">3,125</th><th class="col6">1,5625</th><th class="col7">0,78125</th><th class="col8">0,390625</th><th class="col9">0,1953125</th><th class="col10">0,09765625</th><th class="col11">0,048828125</th><th class="col12">0</th>
 	</tr>
 	</thead>
 	<tr class="row1">
-		<td class="col0">12,5μl</td><td class="col1">Q5 MasterMix 2x</td>
+		<td class="col0">Replicate 1</td><td class="col1 rightalign">	41550</td><td class="col2 rightalign">	28587</td><td class="col3 rightalign">	15976</td><td class="col4 rightalign">	9537</td><td class="col5 rightalign">	5674</td><td class="col6 rightalign">	2712</td><td class="col7 rightalign">	1357</td><td class="col8 rightalign">	639</td><td class="col9 rightalign">	348</td><td class="col10 rightalign">	160</td><td class="col11 rightalign">	91</td><td class="col12 rightalign">	0</td>
 	</tr>
 	<tr class="row2">
-		<td class="col0">1,25μl</td><td class="col1">FW primer (for sgRNAscramble)</td>
+		<td class="col0">Replicate 2</td><td class="col1 rightalign">	42434</td><td class="col2 rightalign">	29060</td><td class="col3 rightalign">	17592</td><td class="col4 rightalign">	10169</td><td class="col5 rightalign">	5250</td><td class="col6 rightalign">	2705</td><td class="col7 rightalign">	1155</td><td class="col8 rightalign">	581</td><td class="col9 rightalign">	302</td><td class="col10 rightalign">	160</td><td class="col11 rightalign">	67</td><td class="col12 rightalign">	11</td>
 	</tr>
 	<tr class="row3">
-		<td class="col0">1,25μl</td><td class="col1">RW primer (olG_118)</td>
+		<td class="col0">Replicate 3</td><td class="col1 rightalign">	42066</td><td class="col2 rightalign">	29363</td><td class="col3 rightalign">	17743</td><td class="col4 rightalign">	9882</td><td class="col5 rightalign">	5015</td><td class="col6 rightalign">	2671</td><td class="col7 rightalign">	1303</td><td class="col8 rightalign">	665</td><td class="col9 rightalign">	314</td><td class="col10 rightalign">	187</td><td class="col11 rightalign">	87</td><td class="col12 rightalign">	0</td>
 	</tr>
 	<tr class="row4">
-		<td class="col0">1μl</td><td class="col1">CAS9-GFP plasmid</td>
+		<td class="col0">Replicate 4</td><td class="col1 rightalign">	40538</td><td class="col2 rightalign">	28766</td><td class="col3 rightalign">	17808</td><td class="col4 rightalign">	9934</td><td class="col5 rightalign">	5127</td><td class="col6 rightalign">	2818</td><td class="col7 rightalign">	1344</td><td class="col8 rightalign">	662</td><td class="col9 rightalign">	366</td><td class="col10 rightalign">	176</td><td class="col11 rightalign">	113</td><td class="col12 rightalign">	13</td>
-	</tr>
-	<tr class="row5">
-		<td class="col0">9μl</td><td class="col1">H2O</td>
 	</tr>
 </table></div>
-<div class='secedit editbutton_table editbutton_173'> </div>
+<div class='secedit editbutton_table editbutton_39'> </div>
 <p>
-PCR program: sgRNA insertion 2.<br/>
+<a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=picture1.png" class="media" title="picture1.png"><img src="/igem2017/lib/exe/fetch.php?w=400&amp;tok=8acd18&amp;media=picture1.png" class="media" alt="" width="400" /></a>
+<a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=picture2.png" class="media" title="picture2.png"><img src="/igem2017/lib/exe/fetch.php?w=400&amp;tok=bfe4be&amp;media=picture2.png" class="media" alt="" width="400" /></a>
 </p>
+<img src="https://static.igem.org/mediawiki/2017/3/3a/T-Freiburg-8_5_2017_Interlab_Study_1.png">
+<img src="https://static.igem.org/mediawiki/2017/e/e7/T-Freiburg-8_5_2017_Interlab_Study_2.png">
-<p>
+</div>
-μl were used for heatshock transformation.<br/>
+<div class='secedit editbutton_section editbutton_38'> </div>
+<h2 class="sectionedit40" id="section060817">06.08.17</h2>
-</p>
+<div class="level2">
 </div>
-<div class='secedit editbutton_section editbutton_172'> </div>
+<div class='secedit editbutton_section editbutton_40'> </div>
-<h3 class="sectionedit174" id="heatshock_transformation_of_xl10gold2">Heatshock transformation of XL10gold</h3>
+<h3 class="sectionedit41" id="pick_colonies_from_interlab_study">pick colonies from Interlab Study</h3>
 <div class="level3">
 <p>
-Heatshock transformation was performed accoarding to the heatshock protocol.<br/>
+- pick 2 colonies from each plate<br/>
+- incubation overnight at 37°C<br/>
 </p>
 </div>
-<div class='secedit editbutton_section editbutton_174'> </div>
+<div class='secedit editbutton_section editbutton_41'> </div>
-<h2 class="sectionedit175" id="section200617">20.06.17</h2>
+<h2 class="sectionedit42" id="section070817">07.08.17</h2>
 <div class="level2">
 </div>
-<div class='secedit editbutton_section editbutton_175'> </div>
+<div class='secedit editbutton_section editbutton_42'> </div>
-<h3 class="sectionedit176" id="test_digest_of_sgen-gibson_clolonies">Test digest of SGEN-Gibson clolonies</h3>
+<h3 class="sectionedit43" id="interlab_study_cell_measurements">Interlab Study cell measurements</h3>
 <div class="level3">
-<div class="table sectionedit177"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0">x</td><td class="col1">80ng DNA</td>
-	</tr>
-	<tr class="row2">
-		<td class="col0">0,2μl</td><td class="col1">NotI</td>
-	</tr>
-	<tr class="row3">
-		<td class="col0">2μl</td><td class="col1"> Cut Smart 10x buffer</td>
-	</tr>
-	<tr class="row4">
-		<td class="col0">to 20μl</td><td class="col1">H2O</td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_177'> </div>
-<p>
-Digest for 45 minutes at 45°C.<br/>
-</p>
 <p>
-Heatshock transformation of colonies 1,2,4,5,7.<br/>
+- measure OD600 of the overnight cultures<br/>
-</p>
+- dilute the bacteria in medium with Chloramphenicol to a final OD = 0.02<br/>
-</div>
+- incubation at 37°C and 220 rpm<br/>
-<div class='secedit editbutton_section editbutton_176'> </div>
-<h3 class="sectionedit178" id="heatshock_transformation_of_dh5α">Heatshock transformation of Dh5α</h3>
-<div class="level3">
-<p>
+- take 500 µl of the cultures after 0h, 2h, 4h and 6h and place samples on ice<br/>
-Heatshock transformation was performed accoarding to the heatshock protocol.<br/>
-</p>
+- transfer four replicates of 100 µl of each sample into a well of a 96-well-plate<br/>
-</div>
+- measure OD600 and fluorescence intensity in the plate reader (settings: excitation: 485 nm; emission: 530/30)<br/>
-<div class='secedit editbutton_section editbutton_178'> </div>
-<h2 class="sectionedit179" id="section210617">21.06.17</h2>
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_179'> </div>
-<h3 class="sectionedit180" id="digest_of_cas9-gfp-sgrna4_3_9_with_dpni">digest of CAS9-GFP-sgRNA4,3,9 with DpnI</h3>
-<div class="level3">
-<div class="table sectionedit181"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0">4μl</td><td class="col1"> Ligation</td>
-	</tr>
-	<tr class="row2">
-		<td class="col0">1μl</td><td class="col1"> Cut Smart 10x buffer</td>
-	</tr>
-	<tr class="row3">
-		<td class="col0">1μl</td><td class="col1"> DpnI</td>
-	</tr>
-	<tr class="row4">
-		<td class="col0">4μl</td><td class="col1">H2O</td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_181'> </div>
-<p>
-Digest for 1 hour at 37°C.<br/>
-μl was used for transformation.<br/>
 </p>
-</div>
-<div class='secedit editbutton_section editbutton_180'> </div>
-<h3 class="sectionedit182" id="heatshock_transformation_of_xl10gold3">Heatshock transformation of XL10gold</h3>
-<div class="level3">
 <p>
-Heatshock transformation was performed accoarding to the heatshock protocol.<br/>
+<strong>Abs600 after 0h:</strong>
 </p>
+<div class="table sectionedit44"><table class="inline">
-</div>
-<div class='secedit editbutton_section editbutton_182'> </div>
-<h2 class="sectionedit183" id="section2106171">21.06.17</h2>
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_183'> </div>
-<h3 class="sectionedit184" id="sequencing">Sequencing</h3>
-<div class="level3">
-<div class="table sectionedit185"><table class="inline">
 	<thead>
 	<tr class="row0">
-		<th class="col0">Plasmid</th><th class="col1">Sequencing Primer</th>
+		<th class="col0"> </th><th class="col1">Neg. Control</th><th class="col2 rightalign">	Pos. Control</th><th class="col3 rightalign">	Device 1</th><th class="col4 rightalign">	Device 2</th><th class="col5 rightalign">	Device 3</th><th class="col6 rightalign">	Device 4</th><th class="col7 rightalign">	Device 5</th><th class="col8 rightalign">	Device 6</th><th class="col9 rightalign">	LB + Chlor (blank)</th>
 	</tr>
 	</thead>
 	<tr class="row1">
-		<td class="col0">SGEN 1</td><td class="col1">Seq SBEN 2</td>
+		<td class="col0 leftalign">Colony 1, Replicate 1	</td><td class="col1 leftalign">0,044	</td><td class="col2 leftalign">0,042	</td><td class="col3 leftalign">0,045	</td><td class="col4 leftalign">0,045	</td><td class="col5 leftalign">0,046	</td><td class="col6 leftalign">0,045	</td><td class="col7 leftalign">0,052	</td><td class="col8 leftalign">0,043	</td><td class="col9">0,041</td>
 	</tr>
 	<tr class="row2">
-		<td class="col0">SGEN 2</td><td class="col1">Seq SBEN 2</td>
+		<td class="col0 leftalign">Colony 1, Replicate 2	</td><td class="col1 leftalign">0,046	</td><td class="col2 leftalign">0,047	</td><td class="col3 leftalign">0,045	</td><td class="col4 leftalign">0,045	</td><td class="col5 leftalign">0,047	</td><td class="col6 leftalign">0,048	</td><td class="col7 leftalign">0,046	</td><td class="col8 leftalign">0,064	</td><td class="col9">0,041</td>
 	</tr>
 	<tr class="row3">
-		<td class="col0">SGEN 3</td><td class="col1">Seq SBEN 2</td>
+		<td class="col0 leftalign">Colony 1, Replicate 3	</td><td class="col1 leftalign">0,044	</td><td class="col2 leftalign">0,045	</td><td class="col3 leftalign">0,047	</td><td class="col4 leftalign">0,044	</td><td class="col5 leftalign">0,054	</td><td class="col6 leftalign">0,046	</td><td class="col7 leftalign">0,045	</td><td class="col8 leftalign">0,045	</td><td class="col9">0,043</td>
 	</tr>
 	<tr class="row4">
-		<td class="col0">SGEN 4</td><td class="col1">Seq SBEN 2</td>
+		<td class="col0 leftalign">Colony 1, Replicate 4	</td><td class="col1 leftalign">0,045	</td><td class="col2 leftalign">0,045	</td><td class="col3 leftalign">0,045	</td><td class="col4 leftalign">0,058	</td><td class="col5 leftalign">0,045	</td><td class="col6 leftalign">0,046	</td><td class="col7 leftalign">0,044	</td><td class="col8 leftalign">0,046	</td><td class="col9">0,046</td>
 	</tr>
 	<tr class="row5">
-		<td class="col0">SGEN 5</td><td class="col1">Seq SBEN 2</td>
+		<td class="col0 leftalign">Colony 2, Replicate 1	</td><td class="col1 leftalign">0,046	</td><td class="col2 leftalign">0,047	</td><td class="col3 leftalign">0,047	</td><td class="col4 leftalign">0,05	</td><td class="col5 leftalign">0,046	</td><td class="col6 leftalign">0,044	</td><td class="col7 leftalign">0,044	</td><td class="col8 leftalign">0,044	</td><td class="col9">0,043</td>
 	</tr>
 	<tr class="row6">
-		<td class="col0">CAS9-GFP-sgRNA20 1</td><td class="col1">olG_117</td>
+		<td class="col0 leftalign">Colony 2, Replicate 2	</td><td class="col1 leftalign">0,046	</td><td class="col2 leftalign">0,05	</td><td class="col3 leftalign">0,045	</td><td class="col4 leftalign">0,045	</td><td class="col5 leftalign">0,045	</td><td class="col6 leftalign">0,046	</td><td class="col7 leftalign">0,05	</td><td class="col8 leftalign">0,046	</td><td class="col9">0,046</td>
 	</tr>
 	<tr class="row7">
-		<td class="col0">CAS9-GFP-sgRNA20 2</td><td class="col1">olG_117</td>
+		<td class="col0 leftalign">Colony 2, Replicate 3	</td><td class="col1 leftalign">0,049	</td><td class="col2 leftalign">0,049	</td><td class="col3 leftalign">0,046	</td><td class="col4 leftalign">0,045	</td><td class="col5 leftalign">0,053	</td><td class="col6 leftalign">0,046	</td><td class="col7 leftalign">0,047	</td><td class="col8 leftalign">0,047	</td><td class="col9">0,044</td>
 	</tr>
 	<tr class="row8">
-		<td class="col0">CAS9-GFP-sgRNA3 1</td><td class="col1">olG_117</td>
+		<td class="col0 leftalign">Colony 2, Replicate 4	</td><td class="col1 leftalign">0,046	</td><td class="col2 leftalign">0,051	</td><td class="col3 leftalign">0,043	</td><td class="col4 leftalign">0,043	</td><td class="col5 leftalign">0,044	</td><td class="col6 leftalign">0,049	</td><td class="col7 leftalign">0,048	</td><td class="col8 leftalign">0,05	</td><td class="col9">0,042</td>
-	</tr>
-	<tr class="row9">
-		<td class="col0">CAS9-GFP-sgRNA9 1</td><td class="col1">olG_117</td>
-	</tr>
-	<tr class="row10">
-		<td class="col0">lenti-CAS-sgRNA1</td><td class="col1">olG_117</td>
-	</tr>
-	<tr class="row11">
-		<td class="col0">lenti-CAS-sgRNA3</td><td class="col1">olG_117</td>
 	</tr>
 </table></div>
-<div class='secedit editbutton_table editbutton_185'> </div>
+<div class='secedit editbutton_table editbutton_44'> </div>
-</div>
+<p>
-<div class='secedit editbutton_section editbutton_184'> </div>
+<strong>Fluorescence intensity after 0h:</strong>
-<h2 class="sectionedit186" id="section220617">22.06.17</h2>
+</p>
-<div class="level2">
+<div class="table sectionedit45"><table class="inline">
-</div>
-<div class='secedit editbutton_section editbutton_186'> </div>
-<h3 class="sectionedit187" id="sdm_pcr_for_shrna_inserts">SDM PCR for shRNA Inserts</h3>
-<div class="level3">
-<div class="table sectionedit188"><table class="inline">
 	<thead>
 	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
+		<th class="col0"> </th><th class="col1 rightalign">	Neg. Control</th><th class="col2 centeralign">	Pos. Control	</th><th class="col3">Device 1</th><th class="col4 centeralign">	Device 2	</th><th class="col5">Device 3</th><th class="col6 centeralign">	Device 4	</th><th class="col7">Device 5</th><th class="col8 centeralign">	Device 6	</th><th class="col9">LB + Chlor (blank)</th>
 	</tr>
 	</thead>
 	<tr class="row1">
-		<td class="col0">10μl</td><td class="col1"> Template</td>
+		<td class="col0 leftalign">Colony 1, Replicate 1	</td><td class="col1 leftalign">30032	</td><td class="col2 leftalign">29468	</td><td class="col3 leftalign">47522	</td><td class="col4 leftalign">30425	</td><td class="col5 leftalign">37202	</td><td class="col6 leftalign">30859	</td><td class="col7 leftalign">38967	</td><td class="col8 leftalign">39848	</td><td class="col9">40167</td>
 	</tr>
 	<tr class="row2">
-		<td class="col0">10μl</td><td class="col1"> Q5 5x buffer</td>
+		<td class="col0 leftalign">Colony 1, Replicate 2	</td><td class="col1 leftalign">38825	</td><td class="col2 leftalign">32134	</td><td class="col3 leftalign">33823	</td><td class="col4 leftalign">37326	</td><td class="col5 leftalign">38129	</td><td class="col6 leftalign">41631	</td><td class="col7 leftalign">37103	</td><td class="col8 leftalign">40926	</td><td class="col9">383</td>
 	</tr>
 	<tr class="row3">
-		<td class="col0">1μl</td><td class="col1"> 10μM dNTPs</td>
+		<td class="col0 leftalign">Colony 1, Replicate 3	</td><td class="col1 leftalign">33426	</td><td class="col2 leftalign">38291	</td><td class="col3 leftalign">40791	</td><td class="col4 leftalign">38829	</td><td class="col5 leftalign">35103	</td><td class="col6 leftalign">35503	</td><td class="col7 leftalign">39082	</td><td class="col8 leftalign">38194	</td><td class="col9">40506</td>
 	</tr>
 	<tr class="row4">
-		<td class="col0">2,5μl</td><td class="col1"> FW primer</td>
+		<td class="col0 leftalign">Colony 1, Replicate 4	</td><td class="col1 leftalign">30451	</td><td class="col2 leftalign">31664	</td><td class="col3 leftalign">40586	</td><td class="col4 leftalign">40661	</td><td class="col5 leftalign">37774	</td><td class="col6 leftalign">40046	</td><td class="col7 leftalign">38716	</td><td class="col8 leftalign">42854	</td><td class="col9">39830</td>
 	</tr>
 	<tr class="row5">
-		<td class="col0">2,5μl</td><td class="col1"> RW primer</td>
+		<td class="col0 leftalign">Colony 2, Replicate 1	</td><td class="col1 leftalign">31429	</td><td class="col2 leftalign">32597	</td><td class="col3 leftalign">33648	</td><td class="col4 leftalign">34085	</td><td class="col5 leftalign">40700	</td><td class="col6 leftalign">31595	</td><td class="col7 leftalign">28914	</td><td class="col8 leftalign">36532	</td><td class="col9">40434</td>
 	</tr>
 	<tr class="row6">
-		<td class="col0">0,5μl</td><td class="col1"> Q5 polymerase</td>
+		<td class="col0 leftalign">Colony 2, Replicate 2	</td><td class="col1 leftalign">39464	</td><td class="col2 leftalign">32361	</td><td class="col3 leftalign">39432	</td><td class="col4 leftalign">40150	</td><td class="col5 leftalign">39543	</td><td class="col6 leftalign">41282	</td><td class="col7 leftalign">38247	</td><td class="col8 leftalign">33320	</td><td class="col9">39231</td>
 	</tr>
 	<tr class="row7">
-		<td class="col0">23,5μl</td><td class="col1"> H20</td>
+		<td class="col0 leftalign">Colony 2, Replicate 3	</td><td class="col1 leftalign">32329	</td><td class="col2 leftalign">31485	</td><td class="col3 leftalign">34366	</td><td class="col4 leftalign">36754	</td><td class="col5 leftalign">39980	</td><td class="col6 leftalign">41296	</td><td class="col7 leftalign">33348	</td><td class="col8 leftalign">33391	</td><td class="col9">39612</td>
 	</tr>
-</table></div>
+	<tr class="row8">
-<div class='secedit editbutton_table editbutton_188'> </div><div class="table sectionedit189"><table class="inline">
+		<td class="col0 leftalign">Colony 2, Replicate 4	</td><td class="col1 leftalign">31225	</td><td class="col2 leftalign">36435	</td><td class="col3 leftalign">37063	</td><td class="col4 leftalign">38806	</td><td class="col5 leftalign">42787	</td><td class="col6 leftalign">40222	</td><td class="col7 leftalign">36968	</td><td class="col8 leftalign">36323	</td><td class="col9">38492</td>
-	<thead>
-	<tr class="row0">
-		<th class="col0">Temp.[°C]</th><th class="col1">Time</th><th class="col2">Cycles</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0">98</td><td class="col1">30 sec.</td><td class="col2"></td>
-	</tr>
-	<tr class="row2">
-		<td class="col0">98</td><td class="col1">10 sec.</td><td class="col2">33</td>
-	</tr>
-	<tr class="row3">
-		<td class="col0">66</td><td class="col1">30 sec.</td><td class="col2">33</td>
-	</tr>
-	<tr class="row4">
-		<td class="col0">72</td><td class="col1">20 sec.</td><td class="col2">33</td>
-	</tr>
-	<tr class="row5">
-		<td class="col0">72</td><td class="col1">2 min.</td><td class="col2">24</td>
 	</tr>
 </table></div>
-<div class='secedit editbutton_table editbutton_189'> </div>
+<div class='secedit editbutton_table editbutton_45'> </div>
-</div>
-<div class='secedit editbutton_section editbutton_187'> </div>
-<h2 class="sectionedit190" id="section240617">24.06.17</h2>
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_190'> </div>
-<h3 class="sectionedit191" id="overnight_culture">overnight culture</h3>
-<div class="level3">
 <p>
-Overnight culture was prepared from glycerol stocks. Plasmid 101, 104,203 of Origene KO-Kit.<br/>
+<strong>Abs600 after 2h:</strong>
 </p>
+<div class="table sectionedit46"><table class="inline">
-</div>
-<div class='secedit editbutton_section editbutton_191'> </div>
-<h2 class="sectionedit192" id="section250617">25.06.17</h2>
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_192'> </div>
-<h3 class="sectionedit193" id="overnight_culture1">overnight culture</h3>
-<div class="level3">
-<p>
-ml for Midi prep was prepared for plasmid 101, 104,203 of Origene KO-Kit.<br/>
-</p>
-</div>
-<div class='secedit editbutton_section editbutton_193'> </div>
-<h2 class="sectionedit194" id="section260617">26.06.17</h2>
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_194'> </div>
-<h3 class="sectionedit195" id="midi_prep1">Midi prep</h3>
-<div class="level3">
-<p>
-Midi prep for plasmid 101, 104,203 of Origene KO-Kit was performed accoarding to the manufacturer&#039;s protocol.<br/>
-</p>
-</div>
-<div class='secedit editbutton_section editbutton_195'> </div>
-<h2 class="sectionedit196" id="section270617">27.06.17</h2>
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_196'> </div>
-<h3 class="sectionedit197" id="heatshock_transformation">Heatshock transformation</h3>
-<div class="level3">
-<p>
-Heatshock transformation of KO-Kit scramble sgRNA was performed accoarding to protocol.<br/>
-</p>
-</div>
-<div class='secedit editbutton_section editbutton_197'> </div>
-<h3 class="sectionedit198" id="colony_pcr_of_lenti-cas9-puro_plasmids">Colony PCR of lenti-CAS9-puro plasmids</h3>
-<div class="level3">
-<div class="table sectionedit199"><table class="inline">
 	<thead>
 	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
+		<th class="col0"> </th><th class="col1 leftalign">Neg. Control	</th><th class="col2">Pos. Control</th><th class="col3 centeralign">	Device 1	</th><th class="col4">Device 2</th><th class="col5 centeralign">	Device 3	</th><th class="col6">Device 4</th><th class="col7 centeralign">	Device 5	</th><th class="col8">Device 6</th><th class="col9 rightalign">	LB + Chlor (blank)</th>
 	</tr>
 	</thead>
 	<tr class="row1">
-		<td class="col0">1 μl</td><td class="col1"> Template</td>
+		<td class="col0 leftalign">Colony 1, Replicate 1	</td><td class="col1 leftalign">0,046	</td><td class="col2 leftalign">0,045	</td><td class="col3 leftalign">0,045	</td><td class="col4 leftalign">0,065	</td><td class="col5 leftalign">0,047	</td><td class="col6 leftalign">0,047	</td><td class="col7 leftalign">0,046	</td><td class="col8 leftalign">0,044	</td><td class="col9">0,041</td>
 	</tr>
 	<tr class="row2">
-		<td class="col0">2 μl</td><td class="col1"> 10x Buffer</td>
+		<td class="col0 leftalign">Colony 1, Replicate 2	</td><td class="col1 leftalign">0,047	</td><td class="col2 leftalign">0,047	</td><td class="col3 leftalign">0,046	</td><td class="col4 leftalign">0,048	</td><td class="col5 leftalign">0,048	</td><td class="col6 leftalign">0,046	</td><td class="col7 leftalign">0,047	</td><td class="col8 leftalign">0,046	</td><td class="col9">0,043</td>
 	</tr>
 	<tr class="row3">
-		<td class="col0">0.4 μl</td><td class="col1"> 10 μM dNTPs</td>
+		<td class="col0 leftalign">Colony 1, Replicate 3	</td><td class="col1 leftalign">0,046	</td><td class="col2 leftalign">0,047	</td><td class="col3 leftalign">0,046	</td><td class="col4 leftalign">0,045	</td><td class="col5 leftalign">0,053	</td><td class="col6 leftalign">0,047	</td><td class="col7 leftalign">0,048	</td><td class="col8 leftalign">0,045	</td><td class="col9">0,044</td>
 	</tr>
 	<tr class="row4">
-		<td class="col0">1 μl</td><td class="col1"> FW primer</td>
+		<td class="col0 leftalign">Colony 1, Replicate 4	</td><td class="col1 leftalign">0,047	</td><td class="col2 leftalign">0,047	</td><td class="col3 leftalign">0,055	</td><td class="col4 leftalign">0,058	</td><td class="col5 leftalign">0,047	</td><td class="col6 leftalign">0,046	</td><td class="col7 leftalign">0,045	</td><td class="col8 leftalign">0,046	</td><td class="col9">0,043</td>
 	</tr>
 	<tr class="row5">
-		<td class="col0">1 μl</td><td class="col1"> RW primer</td>
+		<td class="col0 leftalign">Colony 2, Replicate 1	</td><td class="col1 leftalign">0,078	</td><td class="col2 leftalign">0,048	</td><td class="col3 leftalign">0,048	</td><td class="col4 leftalign">0,049	</td><td class="col5 leftalign">0,048	</td><td class="col6 leftalign">0,047	</td><td class="col7 leftalign">0,046	</td><td class="col8 leftalign">0,047	</td><td class="col9">0,043</td>
 	</tr>
 	<tr class="row6">
-		<td class="col0">0.25 μl</td><td class="col1"> Taq polymerase</td>
+		<td class="col0 leftalign">Colony 2, Replicate 2	</td><td class="col1 leftalign">0,048	</td><td class="col2 leftalign">0,055	</td><td class="col3 leftalign">0,048	</td><td class="col4 leftalign">0,049	</td><td class="col5 leftalign">0,047	</td><td class="col6 leftalign">0,046	</td><td class="col7 leftalign">0,049	</td><td class="col8 leftalign">0,049	</td><td class="col9">0,041</td>
 	</tr>
 	<tr class="row7">
-		<td class="col0">14.35 μl</td><td class="col1"> H20</td>
+		<td class="col0 leftalign">Colony 2, Replicate 3	</td><td class="col1 leftalign">0,047	</td><td class="col2 leftalign">0,048	</td><td class="col3 leftalign">0,048	</td><td class="col4 leftalign">0,048	</td><td class="col5 leftalign">0,047	</td><td class="col6 leftalign">0,049	</td><td class="col7 leftalign">0,048	</td><td class="col8 leftalign">0,05	</td><td class="col9">0,045</td>
 	</tr>
-</table></div>
+	<tr class="row8">
-<div class='secedit editbutton_table editbutton_199'> </div>
+		<td class="col0 leftalign">Colony 2, Replicate 4	</td><td class="col1 leftalign">0,046	</td><td class="col2 leftalign">0,049	</td><td class="col3 leftalign">0,047	</td><td class="col4 leftalign">0,046	</td><td class="col5 leftalign">0,045	</td><td class="col6 leftalign">0,048	</td><td class="col7 leftalign">0,048	</td><td class="col8 leftalign">0,049	</td><td class="col9">0,047</td>
-<p>
-Expected bands: 2,804 bp<br/>
-</p>
-<p>
-<img src="https://static.igem.org/mediawiki/2017/e/ed/T%E2%80%93FREIBURG%E2%80%93465.png" width="500px">
-</p>
-</div>
-<div class='secedit editbutton_section editbutton_198'> </div>
-<h3 class="sectionedit200" id="overnight_digest_of_lenti-cas9">Overnight digest of lenti-Cas9</h3>
-<div class="level3">
-<div class="table sectionedit201"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0">2.5 μl</td><td class="col1"> Plasmid (500ng/µl)</td>
-	</tr>
-	<tr class="row2">
-		<td class="col0">1 µl </td><td class="col1"> BsmBI</td>
-	</tr>
-	<tr class="row3">
-		<td class="col0">5 µl</td><td class="col1"> 3.1 10x buffer</td>
-	</tr>
-	<tr class="row4">
-		<td class="col0">41.5 µl</td><td class="col1"> H2O</td>
 	</tr>
 </table></div>
-<div class='secedit editbutton_table editbutton_201'> </div>
+<div class='secedit editbutton_table editbutton_46'> </div>
 <p>
-Digest overnight at 55°C.<br/>
+<strong>Fluorescence intensity after 2h:</strong>
 </p>
+<div class="table sectionedit47"><table class="inline">
-<p>
-Picture nr. 468<br/>
-</p>
-</div>
-<div class='secedit editbutton_section editbutton_200'> </div>
-<h3 class="sectionedit202" id="colony_pcr_of_cas9-gfp_sgrna1_4_scramble_and_lenti-cas9_sgrna5">Colony PCR of Cas9-GFP sgRNA1/4/scramble and lenti-Cas9 sgRNA5</h3>
-<div class="level3">
-<p>
-Colony PCR was performed according to protocol.<br/>
-For Cas9-GFP: forward primer was singlestrand oligo forward as used to produce double strand oligos for cloning, revese primer 118.<br/>
-For lenti-Cas9: primers 117 and 118.<br/>
-Annealing at 58°C.
-</p>
-</div>
-<div class='secedit editbutton_section editbutton_202'> </div>
-<h2 class="sectionedit203" id="section280617">28.06.17</h2>
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_203'> </div>
-<h3 class="sectionedit204" id="digest_of_lenti-cas9_with_bsmbi">Digest of lenti-Cas9 with BsmBI</h3>
-<div class="level3">
-<div class="table sectionedit205"><table class="inline">
 	<thead>
 	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
+		<th class="col0"> </th><th class="col1 centeralign">	Neg. Control	</th><th class="col2">Pos. Control</th><th class="col3 centeralign">	Device 1	</th><th class="col4">Device 2</th><th class="col5 centeralign">	Device 3	</th><th class="col6">Device 4</th><th class="col7 rightalign">	Device 5</th><th class="col8 centeralign">	Device 6	</th><th class="col9">LB + Chlor (blank)</th>
 	</tr>
 	</thead>
 	<tr class="row1">
-		<td class="col0">1 μl</td><td class="col1"> Template</td>
+		<td class="col0 leftalign">Colony 1, Replicate 1	</td><td class="col1 leftalign">32478	</td><td class="col2 leftalign">41305	</td><td class="col3 leftalign">33618	</td><td class="col4 leftalign">42435	</td><td class="col5 leftalign">34249	</td><td class="col6 leftalign">34205	</td><td class="col7 leftalign">34202	</td><td class="col8 leftalign">34949	</td><td class="col9">29069</td>
 	</tr>
 	<tr class="row2">
-		<td class="col0">2 μl</td><td class="col1"> 10x Buffer</td>
+		<td class="col0 leftalign">Colony 1, Replicate 2	</td><td class="col1 leftalign">34345	</td><td class="col2 leftalign">42706	</td><td class="col3 leftalign">38570	</td><td class="col4 leftalign">43860	</td><td class="col5 leftalign">36547	</td><td class="col6 leftalign">44267	</td><td class="col7 leftalign">45267	</td><td class="col8 leftalign">47187	</td><td class="col9">37138</td>
 	</tr>
 	<tr class="row3">
-		<td class="col0">0.4 μl</td><td class="col1"> 10 μM dNTPs</td>
+		<td class="col0 leftalign">Colony 1, Replicate 3	</td><td class="col1 leftalign">38418	</td><td class="col2 leftalign">39069	</td><td class="col3 leftalign">38866	</td><td class="col4 leftalign">44974	</td><td class="col5 leftalign">36631	</td><td class="col6 leftalign">43986	</td><td class="col7 leftalign">46760	</td><td class="col8 leftalign">39103	</td><td class="col9">39152</td>
 	</tr>
 	<tr class="row4">
-		<td class="col0">1 μl</td><td class="col1"> FW primer</td>
+		<td class="col0 leftalign">Colony 1, Replicate 4	</td><td class="col1 leftalign">36379	</td><td class="col2 leftalign">42391	</td><td class="col3 leftalign">36129	</td><td class="col4 leftalign">45361	</td><td class="col5 leftalign">39003	</td><td class="col6 leftalign">44229	</td><td class="col7 leftalign">32123	</td><td class="col8 leftalign">33637	</td><td class="col9">41422</td>
 	</tr>
 	<tr class="row5">
-		<td class="col0">1 μl</td><td class="col1"> RW primer</td>
+		<td class="col0 leftalign">Colony 2, Replicate 1	</td><td class="col1 leftalign">36339	</td><td class="col2 leftalign">36658	</td><td class="col3 leftalign">36464	</td><td class="col4 leftalign">35639	</td><td class="col5 leftalign">43270	</td><td class="col6 leftalign">42121	</td><td class="col7 leftalign">34385	</td><td class="col8 leftalign">44627	</td><td class="col9">40854</td>
 	</tr>
 	<tr class="row6">
-		<td class="col0">0.25 μl</td><td class="col1"> Taq polymerase</td>
+		<td class="col0 leftalign">Colony 2, Replicate 2	</td><td class="col1 leftalign">40618	</td><td class="col2 leftalign">43596	</td><td class="col3 leftalign">36428	</td><td class="col4 leftalign">44147	</td><td class="col5 leftalign">44768	</td><td class="col6 leftalign">43257	</td><td class="col7 leftalign">45151	</td><td class="col8 leftalign">46402	</td><td class="col9">39718</td>
 	</tr>
 	<tr class="row7">
-		<td class="col0">14.35 μl</td><td class="col1"> H20</td>
+		<td class="col0 leftalign">Colony 2, Replicate 3	</td><td class="col1 leftalign">39505	</td><td class="col2 leftalign">36517	</td><td class="col3 leftalign">43094	</td><td class="col4 leftalign">44747	</td><td class="col5 leftalign">36790	</td><td class="col6 leftalign">40034	</td><td class="col7 leftalign">44931	</td><td class="col8 leftalign">40597	</td><td class="col9">39712</td>
 	</tr>
-</table></div>
+	<tr class="row8">
-<div class='secedit editbutton_table editbutton_205'> </div>
+		<td class="col0 leftalign">Colony 2, Replicate 4	</td><td class="col1 leftalign">36751	</td><td class="col2 leftalign">41857	</td><td class="col3 leftalign">35023	</td><td class="col4 leftalign">41187	</td><td class="col5 leftalign">35201	</td><td class="col6 leftalign">43423	</td><td class="col7 leftalign">32927	</td><td class="col8 leftalign">35063	</td><td class="col9">37786</td>
-<p>
-Expected bands: 2,804 bp<br/>
-</p>
-<p>
-Picture no. 465<br/>
-</p>
-</div>
-<div class='secedit editbutton_section editbutton_204'> </div>
-<h3 class="sectionedit206" id="overnight_digest_of_lenti-cas91">Overnight digest of lenti-Cas9</h3>
-<div class="level3">
-<div class="table sectionedit207"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0">2.5 μl</td><td class="col1"> Plasmid (500ng/µl)</td>
-	</tr>
-	<tr class="row2">
-		<td class="col0">1 µl </td><td class="col1"> BsmBI</td>
-	</tr>
-	<tr class="row3">
-		<td class="col0">5 µl</td><td class="col1"> 3.1 10x buffer</td>
-	</tr>
-	<tr class="row4">
-		<td class="col0">41.5 µl</td><td class="col1"> H2O</td>
 	</tr>
 </table></div>
-<div class='secedit editbutton_table editbutton_207'> </div>
+<div class='secedit editbutton_table editbutton_47'> </div>
 <p>
-Digested for 2h at 55°C.<br/>
+<strong>Abs600 after 4h:</strong>
-Gel overnight 2h undigested, picture nr. 468<br/>
-</p>
-</div>
-<div class='secedit editbutton_section editbutton_206'> </div>
-<h3 class="sectionedit208" id="miniprep_of_lenti-cas9">Miniprep of lenti-Cas9</h3>
-<div class="level3">
-<p>
-Miniprep was performed according to the manufacturer&#039;s protocol.
-</p>
-</div>
-<div class='secedit editbutton_section editbutton_208'> </div>
-<h3 class="sectionedit209" id="pcr_of_knockdown_inserts">PCR of knockdown inserts</h3>
-<div class="level3">
-<p>
-Templates (97meres): olG17_112 to olG17_116.<br/>
 </p>
-<div class="table sectionedit210"><table class="inline">
+<div class="table sectionedit48"><table class="inline">
 	<thead>
 	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
+		<th class="col0"> </th><th class="col1 centeralign">	Neg. Control	</th><th class="col2 leftalign">Pos. Control	</th><th class="col3 leftalign">Device 1	</th><th class="col4">Device 2</th><th class="col5 centeralign">	Device 3	</th><th class="col6">Device 4</th><th class="col7 rightalign">	Device 5</th><th class="col8 rightalign">	Device 6</th><th class="col9 rightalign">	LB + Chlor (blank)</th>
 	</tr>
 	</thead>
 	<tr class="row1">
-		<td class="col0"> 10.0 µl </td><td class="col1"> 5x Q5Bfr </td>
+		<td class="col0 leftalign">Colony 1, Replicate 1	</td><td class="col1 leftalign">0,056	</td><td class="col2 leftalign">0,055	</td><td class="col3 leftalign">0,055	</td><td class="col4 leftalign">0,052	</td><td class="col5 leftalign">0,058	</td><td class="col6 leftalign">0,052	</td><td class="col7 leftalign">0,053	</td><td class="col8 leftalign">0,046	</td><td class="col9">0,042</td>
 	</tr>
 	<tr class="row2">
-		<td class="col0"> 1.0 µl </td><td class="col1"> dNTPs (10mM) </td>
+		<td class="col0 leftalign">Colony 1, Replicate 2	</td><td class="col1 leftalign">0,054	</td><td class="col2 leftalign">0,056	</td><td class="col3 leftalign">0,054	</td><td class="col4 leftalign">0,052	</td><td class="col5 leftalign">0,055	</td><td class="col6 leftalign">0,054	</td><td class="col7 leftalign">0,055	</td><td class="col8 leftalign">0,048	</td><td class="col9">0,041</td>
 	</tr>
 	<tr class="row3">
-		<td class="col0"> 2.5 µl </td><td class="col1"> 5&#039;mirEXhoIfw (10 µM) </td>
+		<td class="col0 leftalign">Colony 1, Replicate 3	</td><td class="col1 leftalign">0,055	</td><td class="col2 leftalign">0,061	</td><td class="col3 leftalign">0,055	</td><td class="col4 leftalign">0,052	</td><td class="col5 leftalign">0,057	</td><td class="col6 leftalign">0,052	</td><td class="col7 leftalign">0,055	</td><td class="col8 leftalign">0,049	</td><td class="col9">0,043</td>
 	</tr>
 	<tr class="row4">
-		<td class="col0"> 2.5 µl </td><td class="col1"> 3&#039;mirEEcoRIrv (10 µM) </td>
+		<td class="col0 leftalign">Colony 1, Replicate 4	</td><td class="col1 leftalign">0,055	</td><td class="col2 leftalign">0,056	</td><td class="col3 leftalign">0,05	</td><td class="col4 leftalign">0,053	</td><td class="col5 leftalign">0,053	</td><td class="col6 leftalign">0,052	</td><td class="col7 leftalign">0,05	</td><td class="col8 leftalign">0,049	</td><td class="col9">0,043</td>
 	</tr>
 	<tr class="row5">
-		<td class="col0"> 2.0 µl </td><td class="col1"> template (0.01 ng/µl) </td>
+		<td class="col0 leftalign">Colony 2, Replicate 1	</td><td class="col1 leftalign">0,053	</td><td class="col2 leftalign">0,059	</td><td class="col3 leftalign">0,057	</td><td class="col4 leftalign">0,061	</td><td class="col5 leftalign">0,053	</td><td class="col6 leftalign">0,059	</td><td class="col7 leftalign">0,053	</td><td class="col8 leftalign">0,059	</td><td class="col9">0,043</td>
 	</tr>
 	<tr class="row6">
-		<td class="col0"> 0.5 µl </td><td class="col1"> Q5 Pol </td>
+		<td class="col0 leftalign">Colony 2, Replicate 2	</td><td class="col1 leftalign">0,054	</td><td class="col2 leftalign">0,056	</td><td class="col3 leftalign">0,058	</td><td class="col4 leftalign">0,062	</td><td class="col5 leftalign">0,053	</td><td class="col6 leftalign">0,059	</td><td class="col7 leftalign">0,055	</td><td class="col8 leftalign">0,059	</td><td class="col9">0,043</td>
 	</tr>
 	<tr class="row7">
-		<td class="col0"> 31.5 µl </td><td class="col1"> H2O </td>
+		<td class="col0 leftalign">Colony 2, Replicate 3	</td><td class="col1 leftalign">0,054	</td><td class="col2 leftalign">0,056	</td><td class="col3 leftalign">0,058	</td><td class="col4 leftalign">0,063	</td><td class="col5 leftalign">0,057	</td><td class="col6 leftalign">0,059	</td><td class="col7 leftalign">0,055	</td><td class="col8 leftalign">0,06	</td><td class="col9">0,043</td>
 	</tr>
-</table></div>
+	<tr class="row8">
-<div class='secedit editbutton_table editbutton_210'> </div>
+		<td class="col0 leftalign">Colony 2, Replicate 4	</td><td class="col1 leftalign">0,054	</td><td class="col2 leftalign">0,056	</td><td class="col3 leftalign">0,056	</td><td class="col4 leftalign">0,058	</td><td class="col5 leftalign">0,049	</td><td class="col6 leftalign">0,058	</td><td class="col7 leftalign">0,056	</td><td class="col8 leftalign">0,061	</td><td class="col9">0,042</td>
-<p>
-PCR program as described on 22.06.17.<br/>
-Purification with PCR purification kit according to the manufacturer&#039;s protocol (Qiagen), elution in 30 µl H2O.<br/>
-</p>
-</div>
-<div class='secedit editbutton_section editbutton_209'> </div>
-<h3 class="sectionedit211" id="digest_of_lenti-cas9_with_bsmbi1">Digest of lenti-Cas9 with BsmbI</h3>
-<div class="level3">
-<div class="table sectionedit212"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0">2.5 μl</td><td class="col1"> Plasmid (500ng/µl)</td>
-	</tr>
-	<tr class="row2">
-		<td class="col0">1 µl </td><td class="col1"> BsmBI</td>
-	</tr>
-	<tr class="row3">
-		<td class="col0">5 µl</td><td class="col1"> 3.1 10x buffer</td>
-	</tr>
-	<tr class="row4">
-		<td class="col0">41.5 µl</td><td class="col1"> H2O</td>
 	</tr>
 </table></div>
-<div class='secedit editbutton_table editbutton_212'> </div>
+<div class='secedit editbutton_table editbutton_48'> </div>
 <p>
-Digested at 55°C for 2 h.<br/>
+<strong>Fluorescence intensity after 4h:
+</strong>
 </p>
-<div class="table sectionedit213"><table class="inline">
+<div class="table sectionedit49"><table class="inline">
 	<thead>
 	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
+		<th class="col0"> </th><th class="col1 centeralign">	Neg. Control	</th><th class="col2">Pos. Control</th><th class="col3 centeralign">	Device 1	</th><th class="col4">Device 2</th><th class="col5 centeralign">	Device 3	</th><th class="col6">Device 4</th><th class="col7 rightalign">	Device 5</th><th class="col8 rightalign">	Device 6</th><th class="col9 rightalign">	LB + Chlor (blank)</th>
 	</tr>
 	</thead>
 	<tr class="row1">
-		<td class="col0">25 μl</td><td class="col1"> Plasmid (500ng/µl)</td>
+		<td class="col0 leftalign">Colony 1, Replicate 1	</td><td class="col1 leftalign">39398	</td><td class="col2 leftalign">41211	</td><td class="col3 leftalign">43472	</td><td class="col4 leftalign">34629	</td><td class="col5 leftalign">40024	</td><td class="col6 leftalign">37129	</td><td class="col7 leftalign">34265	</td><td class="col8 leftalign">40517	</td><td class="col9">38102</td>
 	</tr>
 	<tr class="row2">
-		<td class="col0">1 µl </td><td class="col1"> BsmBI</td>
+		<td class="col0 leftalign">Colony 1, Replicate 2	</td><td class="col1 leftalign">39957	</td><td class="col2 leftalign">41499	</td><td class="col3 leftalign">44252	</td><td class="col4 leftalign">43313	</td><td class="col5 leftalign">44778	</td><td class="col6 leftalign">36906	</td><td class="col7 leftalign">43865	</td><td class="col8 leftalign">42985	</td><td class="col9">38644</td>
 	</tr>
 	<tr class="row3">
-		<td class="col0">5 µl</td><td class="col1"> 3.1 10x buffer</td>
+		<td class="col0 leftalign">Colony 1, Replicate 3	</td><td class="col1 leftalign">39198	</td><td class="col2 leftalign">41382	</td><td class="col3 leftalign">35548	</td><td class="col4 leftalign">45574	</td><td class="col5 leftalign">47239	</td><td class="col6 leftalign">39866	</td><td class="col7 leftalign">44132	</td><td class="col8 leftalign">44431	</td><td class="col9">37943</td>
 	</tr>
 	<tr class="row4">
-		<td class="col0">19 µl</td><td class="col1"> H2O</td>
+		<td class="col0 leftalign">Colony 1, Replicate 4	</td><td class="col1 leftalign">35352	</td><td class="col2 leftalign">41590	</td><td class="col3 leftalign">26466	</td><td class="col4 leftalign">44768	</td><td class="col5 leftalign">45637	</td><td class="col6 leftalign">44736	</td><td class="col7 leftalign">29248	</td><td class="col8 leftalign">37825	</td><td class="col9">38618</td>
 	</tr>
-</table></div>
+	<tr class="row5">
-<div class='secedit editbutton_table editbutton_213'> </div>
+		<td class="col0 leftalign">Colony 2, Replicate 1	</td><td class="col1 leftalign">41468	</td><td class="col2 leftalign">42577	</td><td class="col3 leftalign">34717	</td><td class="col4 leftalign">39284	</td><td class="col5 leftalign">40334	</td><td class="col6 leftalign">42558	</td><td class="col7 leftalign">46026	</td><td class="col8 leftalign">41271	</td><td class="col9">39053</td>
-<p>
-Digested at 55°C overnight.<br/>
-</p>
-<div class="table sectionedit214"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
 	</tr>
-	</thead>
+	<tr class="row6">
-	<tr class="row1">
+		<td class="col0 leftalign">Colony 2, Replicate 2	</td><td class="col1 leftalign">43892	</td><td class="col2 leftalign">40143	</td><td class="col3 leftalign">36619	</td><td class="col4 leftalign">42028	</td><td class="col5 leftalign">41878	</td><td class="col6 leftalign">43940	</td><td class="col7 leftalign">38303	</td><td class="col8 leftalign">47567	</td><td class="col9">37978</td>
-		<td class="col0">2.5 μl</td><td class="col1"> Plasmid (500ng/µl)</td>
 	</tr>
-	<tr class="row2">
+	<tr class="row7">
-		<td class="col0">1 µl </td><td class="col1"> BsmBI</td>
+		<td class="col0 leftalign">Colony 2, Replicate 3	</td><td class="col1 leftalign">43598	</td><td class="col2 leftalign">43486	</td><td class="col3 leftalign">44578	</td><td class="col4 leftalign">44449	</td><td class="col5 leftalign">44828	</td><td class="col6 leftalign">44235	</td><td class="col7 leftalign">42442	</td><td class="col8 leftalign">37273	</td><td class="col9">39116</td>
 	</tr>
-	<tr class="row3">
+	<tr class="row8">
-		<td class="col0">5 µl</td><td class="col1"> 3.1 10x buffer</td>
+		<td class="col0 leftalign">Colony 2, Replicate 4	</td><td class="col1 leftalign">43048	</td><td class="col2 leftalign">43193	</td><td class="col3 leftalign">42421	</td><td class="col4 leftalign">38871	</td><td class="col5 leftalign">34908	</td><td class="col6 leftalign">41518	</td><td class="col7 leftalign">43876	</td><td class="col8 leftalign">44582	</td><td class="col9">37838</td>
-	</tr>
-	<tr class="row4">
-		<td class="col0">41.5 µl</td><td class="col1"> H2O</td>
 	</tr>
 </table></div>
-<div class='secedit editbutton_table editbutton_214'> </div>
+<div class='secedit editbutton_table editbutton_49'> </div>
 <p>
-Digested at 37°C overnight.<br/>
+<strong>Abs600 after 6h:</strong>
 </p>
+<div class="table sectionedit50"><table class="inline">
-</div>
-<div class='secedit editbutton_section editbutton_211'> </div>
-<h2 class="sectionedit215" id="section290617">29.06.17</h2>
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_215'> </div>
-<h3 class="sectionedit216" id="digest_of_lenti-cas9_with_bambi">Digest of lenti-Cas9 with BamBI</h3>
-<div class="level3">
-<div class="table sectionedit217"><table class="inline">
 	<thead>
 	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
+		<th class="col0"> </th><th class="col1 leftalign">Neg. Control	</th><th class="col2">Pos. Control</th><th class="col3 centeralign">	Device 1	</th><th class="col4">Device 2</th><th class="col5 rightalign">	Device 3</th><th class="col6 rightalign">	Device 4</th><th class="col7 rightalign">	Device 5</th><th class="col8 rightalign">	Device 6</th><th class="col9 rightalign">	LB + Chlor (blank)</th>
 	</tr>
 	</thead>
 	<tr class="row1">
-		<td class="col0">2.5 μl</td><td class="col1"> Plasmid (500ng/µl)</td>
+		<td class="col0 leftalign">Colony 1, Replicate 1	</td><td class="col1 leftalign">0,098	</td><td class="col2 leftalign">0,093	</td><td class="col3 leftalign">0,084	</td><td class="col4 leftalign">0,084	</td><td class="col5 leftalign">0,087	</td><td class="col6 leftalign">0,078	</td><td class="col7 leftalign">0,083	</td><td class="col8 leftalign">0,062	</td><td class="col9">0,048</td>
 	</tr>
 	<tr class="row2">
-		<td class="col0">1 µl </td><td class="col1"> BsmBI</td>
+		<td class="col0 leftalign">Colony 1, Replicate 2	</td><td class="col1 leftalign">0,09	</td><td class="col2 leftalign">0,094	</td><td class="col3 leftalign">0,082	</td><td class="col4 leftalign">0,076	</td><td class="col5 leftalign">0,089	</td><td class="col6 leftalign">0,083	</td><td class="col7 leftalign">0,091	</td><td class="col8 leftalign">0,064	</td><td class="col9">0,041</td>
 	</tr>
 	<tr class="row3">
-		<td class="col0">5 µl</td><td class="col1"> 3.1 10x buffer</td>
+		<td class="col0 leftalign">Colony 1, Replicate 3	</td><td class="col1 leftalign">0,091	</td><td class="col2 leftalign">0,09	</td><td class="col3 leftalign">0,92	</td><td class="col4 leftalign">0,082	</td><td class="col5 leftalign">0,089	</td><td class="col6 leftalign">0,08	</td><td class="col7 leftalign">0,086	</td><td class="col8 leftalign">0,066	</td><td class="col9">0,043</td>
 	</tr>
 	<tr class="row4">
-		<td class="col0">41.5 µl</td><td class="col1"> H2O</td>
+		<td class="col0 leftalign">Colony 1, Replicate 4	</td><td class="col1 leftalign">0,086	</td><td class="col2 leftalign">0,084	</td><td class="col3 leftalign">0,09	</td><td class="col4 leftalign">0,079	</td><td class="col5 leftalign">0,086	</td><td class="col6 leftalign">0,078	</td><td class="col7 leftalign">0,084	</td><td class="col8 leftalign">0,063	</td><td class="col9">0,043</td>
 	</tr>
-</table></div>
+	<tr class="row5">
-<div class='secedit editbutton_table editbutton_217'> </div>
+		<td class="col0 leftalign">Colony 2, Replicate 1	</td><td class="col1 leftalign">0,081	</td><td class="col2 leftalign">0,093	</td><td class="col3 leftalign">0,094	</td><td class="col4 leftalign">0,076	</td><td class="col5 leftalign">0,075	</td><td class="col6 leftalign">0,097	</td><td class="col7 leftalign">0,087	</td><td class="col8 leftalign">0,097	</td><td class="col9">0,043</td>
-<p>
-Digested at 55°C for 2 h.
-<img src="https://static.igem.org/mediawiki/2017/f/f7/T%E2%80%93FREIBURG%E2%80%93471.png" width="500px">
-</p>
-</div>
-<div class='secedit editbutton_section editbutton_216'> </div>
-<h3 class="sectionedit218" id="miniprep_of_lenti-cas91">Miniprep of lenti-CAS9</h3>
-<div class="level3">
-<p>
-No. 1 grew at 30°C, No. 2&amp;3 at 37°C.<br/>
-Miniprep was performed according to manufacturer&#039;s protocol.<br/>
-µl of mini was loaded on gel (picture no. 473).
-</p>
-</div>
-<div class='secedit editbutton_section editbutton_218'> </div>
-<h2 class="sectionedit219" id="section300617">30.06.17</h2>
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_219'> </div>
-<h3 class="sectionedit220" id="gel_extraction_of_digested_lenti-cas9">Gel extraction of digested lenti-Cas9</h3>
-<div class="level3">
-<p>
-Gel extraction was performed according to manufacturer&#039;s protocol.
-</p>
-</div>
-<div class='secedit editbutton_section editbutton_220'> </div>
-              </div>
-            <button class="accordion">July</button>
-        <div id="vanish" class="panel panel-default">
-<h2 class="sectionedit221" id="section010717">01.07.17</h2>
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_221'> </div>
-<h3 class="sectionedit222" id="dephosphorylation_of_backbone">Dephosphorylation of backbone</h3>
-<div class="level3">
-<p>
-Was performed according to manufacturer&#039;s  protocol (Fast AP).<br/>
-</p>
-</div>
-<div class='secedit editbutton_section editbutton_222'> </div>
-<h3 class="sectionedit223" id="ligating_sgrna_into_lenti-cas9_backbone">Ligating sgRNA into lenti-Cas9 backbone</h3>
-<div class="level3">
-<p>
-Annealed sgRNA oligos: 1, 3, 4, 5, 9, 20<br/>
-</p>
-<div class="table sectionedit224"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
 	</tr>
-	</thead>
+	<tr class="row6">
-	<tr class="row1">
+		<td class="col0 leftalign">Colony 2, Replicate 2	</td><td class="col1 leftalign">0,079	</td><td class="col2 leftalign">0,092	</td><td class="col3 leftalign">0,091	</td><td class="col4 leftalign">0,099	</td><td class="col5 leftalign">0,073	</td><td class="col6 leftalign">0,095	</td><td class="col7 leftalign">0,085	</td><td class="col8 leftalign">0,107	</td><td class="col9">0,043</td>
-		<td class="col0"> 7 µl </td><td class="col1"> backbone (9.3 ng/µl)</td>
 	</tr>
-	<tr class="row2">
+	<tr class="row7">
-		<td class="col0"> 1 µl </td><td class="col1"> annealed sgRNA oligo (50 nM) </td>
+		<td class="col0 leftalign">Colony 2, Replicate 3	</td><td class="col1 leftalign">0,08	</td><td class="col2 leftalign">0,086	</td><td class="col3 leftalign">0,101	</td><td class="col4 leftalign">0,13	</td><td class="col5 leftalign">0,073	</td><td class="col6 leftalign">0,1	</td><td class="col7 leftalign">0,088	</td><td class="col8 leftalign">0,107	</td><td class="col9">0,042</td>
 	</tr>
-	<tr class="row3">
+	<tr class="row8">
-		<td class="col0"> 1 µl </td><td class="col1"> 10x T4 buffer </td>
+		<td class="col0 leftalign">Colony 2, Replicate 4	</td><td class="col1 leftalign">0,078	</td><td class="col2 leftalign">0,092	</td><td class="col3 leftalign">0,089	</td><td class="col4 leftalign">0,108	</td><td class="col5 leftalign">0,073	</td><td class="col6 leftalign">0,093	</td><td class="col7 leftalign">0,09	</td><td class="col8 leftalign">0,105	</td><td class="col9">0,042</td>
-	</tr>
-	<tr class="row4">
-		<td class="col0"> 1µl </td><td class="col1"> T4 </td>
 	</tr>
 </table></div>
-<div class='secedit editbutton_table editbutton_224'> </div>
+<div class='secedit editbutton_table editbutton_50'> </div>
-</div>
-<div class='secedit editbutton_section editbutton_223'> </div>
-<h3 class="sectionedit225" id="transformation_of_ligation">Transformation of ligation</h3>
-<div class="level3">
 <p>
-sgRNAs: 1, 3, 4, 5, 9, 20 &amp; scramble of KO kit
+<strong>Fluorescence intensity after 6h:</strong>
-Transformation was performed according to heat shock protocol.<br/>
-–&gt; Only colonies containing sgRNAscramble were obtained.
 </p>
+<div class="table sectionedit51"><table class="inline">
-</div>
-<div class='secedit editbutton_section editbutton_225'> </div>
-<h2 class="sectionedit226" id="section020717">02.07.17</h2>
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_226'> </div>
-<h3 class="sectionedit227" id="digest_of_lenti-cas9_with_bsmbi2">Digest of lenti-Cas9 with BSmbI</h3>
-<div class="level3">
-<div class="table sectionedit228"><table class="inline">
 	<thead>
 	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
+		<th class="col0"> </th><th class="col1 rightalign">	Neg. Control</th><th class="col2 centeralign">	Pos. Control	</th><th class="col3">Device 1</th><th class="col4 centeralign">	Device 2	</th><th class="col5">Device 3</th><th class="col6 rightalign">	Device 4</th><th class="col7 rightalign">	Device 5</th><th class="col8 centeralign">	Device 6	</th><th class="col9">LB + Chlor (blank)</th>
 	</tr>
 	</thead>
 	<tr class="row1">
-		<td class="col0">2.5 μl</td><td class="col1"> Plasmid (500ng/µl)</td>
+		<td class="col0 leftalign">Colony 1, Replicate 1	</td><td class="col1 leftalign">44819	</td><td class="col2 leftalign">43197	</td><td class="col3 leftalign">34990	</td><td class="col4 leftalign">39952	</td><td class="col5 leftalign">35312	</td><td class="col6 leftalign">44026	</td><td class="col7 leftalign">44108	</td><td class="col8 leftalign">34395	</td><td class="col9">41891</td>
 	</tr>
 	<tr class="row2">
-		<td class="col0">1 µl </td><td class="col1"> BsmBI</td>
+		<td class="col0 leftalign">Colony 1, Replicate 2	</td><td class="col1 leftalign">35745	</td><td class="col2 leftalign">41624	</td><td class="col3 leftalign">42712	</td><td class="col4 leftalign">36773	</td><td class="col5 leftalign">38191	</td><td class="col6 leftalign">39327	</td><td class="col7 leftalign">36222	</td><td class="col8 leftalign">38684	</td><td class="col9">44486</td>
 	</tr>
 	<tr class="row3">
-		<td class="col0">5 µl</td><td class="col1"> 3.1 10x buffer</td>
+		<td class="col0 leftalign">Colony 1, Replicate 3	</td><td class="col1 leftalign">43503	</td><td class="col2 leftalign">43470	</td><td class="col3 leftalign">38934	</td><td class="col4 leftalign">39338	</td><td class="col5 leftalign">39338	</td><td class="col6 leftalign">47418	</td><td class="col7 leftalign">46563	</td><td class="col8 leftalign">41520	</td><td class="col9">40216</td>
 	</tr>
 	<tr class="row4">
-		<td class="col0">41.5 µl</td><td class="col1"> H2O</td>
+		<td class="col0 leftalign">Colony 1, Replicate 4	</td><td class="col1 leftalign">40336	</td><td class="col2 leftalign">39507	</td><td class="col3 leftalign">37827	</td><td class="col4 leftalign">41517	</td><td class="col5 leftalign">41517	</td><td class="col6 leftalign">46271	</td><td class="col7 leftalign">47662	</td><td class="col8 leftalign">42298	</td><td class="col9">38353</td>
 	</tr>
-</table></div>
+	<tr class="row5">
-<div class='secedit editbutton_table editbutton_228'> </div>
+		<td class="col0 leftalign">Colony 2, Replicate 1	</td><td class="col1 leftalign">34982	</td><td class="col2 leftalign">34672	</td><td class="col3 leftalign">46394	</td><td class="col4 leftalign">18665	</td><td class="col5 leftalign">18665	</td><td class="col6 leftalign">36165	</td><td class="col7 leftalign">36374	</td><td class="col8 leftalign">40293	</td><td class="col9">40211</td>
-<p>
-Digested at 55°C for 2 h.
-</p>
-</div>
-<div class='secedit editbutton_section editbutton_227'> </div>
-<h3 class="sectionedit229" id="ligating_shrna_into_lenti-cas9_backbone">Ligating shRNA into lenti-Cas9 backbone</h3>
-<div class="level3">
-<p>
-sgRNAs: 1, 3, 4, 5, 9, 20<br/>
-</p>
-<div class="table sectionedit230"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
 	</tr>
-	</thead>
+	<tr class="row6">
-	<tr class="row1">
+		<td class="col0 leftalign">Colony 2, Replicate 2	</td><td class="col1 leftalign">43791	</td><td class="col2 leftalign">37273	</td><td class="col3 leftalign">44467	</td><td class="col4 leftalign">41079	</td><td class="col5 leftalign">41079	</td><td class="col6 leftalign">41516	</td><td class="col7 leftalign">44327	</td><td class="col8 leftalign">38827	</td><td class="col9">38014</td>
-		<td class="col0"> 7 µl </td><td class="col1"> backbone (9.3 ng/µl)</td>
 	</tr>
-	<tr class="row2">
+	<tr class="row7">
-		<td class="col0"> 1 µl </td><td class="col1"> annealed sgRNA oligo (50 nM) </td>
+		<td class="col0 leftalign">Colony 2, Replicate 3	</td><td class="col1 leftalign">39248	</td><td class="col2 leftalign">40869	</td><td class="col3 leftalign">38169	</td><td class="col4 leftalign">48933	</td><td class="col5 leftalign">48933	</td><td class="col6 leftalign">44903	</td><td class="col7 leftalign">41210	</td><td class="col8 leftalign">38643	</td><td class="col9">40437</td>
 	</tr>
-	<tr class="row3">
+	<tr class="row8">
-		<td class="col0"> 1 µl </td><td class="col1"> 10x T4 buffer </td>
+		<td class="col0 leftalign">Colony 2, Replicate 4	</td><td class="col1 leftalign">38078	</td><td class="col2 leftalign">38846	</td><td class="col3 leftalign">41889	</td><td class="col4 leftalign">49892	</td><td class="col5 leftalign">49892	</td><td class="col6 leftalign">42376	</td><td class="col7 leftalign">46132	</td><td class="col8 leftalign">38525	</td><td class="col9">39913</td>
-	</tr>
-	<tr class="row4">
-		<td class="col0"> 1µl </td><td class="col1"> T4 </td>
 	</tr>
 </table></div>
-<div class='secedit editbutton_table editbutton_230'> </div>
+<div class='secedit editbutton_table editbutton_51'> </div>
-<p>
-Ligation was performed at 16°C overnight.
-</p>
 </div>
-<div class='secedit editbutton_section editbutton_229'> </div>
+<div class='secedit editbutton_section editbutton_43'> </div>
-<h2 class="sectionedit231" id="section030717">03.07.17</h2>
+<h2 class="sectionedit52" id="section1907270717">19.08.-27.08.17</h2>
 <div class="level2">
 </div>
-<div class='secedit editbutton_section editbutton_231'> </div>
+<div class='secedit editbutton_section editbutton_52'> </div>
-<h3 class="sectionedit232" id="transformation_of_ligation1">Transformation of ligation</h3>
+<h3 class="sectionedit53" id="ph_change_over_time_in_incubator_with_rpmi1640_and_acid">pH change over time in incubator with RPMI1640 and acid</h3>
 <div class="level3">
+<div class="table sectionedit54"><table class="inline">
-<p>
-sgRNAscramble served as control.<br/>
-Transformation was performed according to heat shock protocol.<br/>
-</p>
-</div>
-<div class='secedit editbutton_section editbutton_232'> </div>
-<h2 class="sectionedit233" id="section040717">04.07.17</h2>
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_233'> </div>
-<h3 class="sectionedit234" id="gel_extraction_and_ligation">Gel extraction and ligation</h3>
-<div class="level3">
-<div class="table sectionedit235"><table class="inline">
 	<thead>
 	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
+		<th class="col0">composition</th><th class="col1">start pH</th><th class="col2">incubation time</th><th class="col3">trend</th>
 	</tr>
 	</thead>
 	<tr class="row1">
-		<td class="col0"> 12 µl </td><td class="col1"> backbone </td>
+		<td class="col0">2ml RPMI1640</td><td class="col1">7.14</td><td class="col2">93 min</td><td class="col3"><img style="max-height: 300px" src="https://static.igem.org/mediawiki/2017/6/69/T-Freiburg-7_19_2017_pH_change_1.png"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=no_acid_1.png" class="media" title="no_acid_1.png"><img src="/igem2017/lib/exe/fetch.php?w=200&amp;tok=2c37ff&amp;media=no_acid_1.png" class="mediacenter" alt="" width="200" /></a></td>
 	</tr>
 	<tr class="row2">
-		<td class="col0"> 1 µl </td><td class="col1"> sgRNA 1 &amp; 9 </td>
+		<td class="col0">2ml RPMI1640</td><td class="col1">7.16</td><td class="col2">67 min</td><td class="col3"><img style="max-height: 300px" src="https://static.igem.org/mediawiki/2017/5/52/T-Freiburg-7_19_2017_pH_change_2.png"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=no_acid_2.png" class="media" title="no_acid_2.png"><img src="/igem2017/lib/exe/fetch.php?w=200&amp;tok=794dc5&amp;media=no_acid_2.png" class="mediacenter" alt="" width="200" /></a></td>
 	</tr>
 	<tr class="row3">
-		<td class="col0"> 1 µl </td><td class="col1"> 10x T4 buffer </td>
+		<td class="col0">2ml RPMI1640</td><td class="col1">7.15</td><td class="col2">104 min</td><td class="col3"><img style="max-height: 300px" src="https://static.igem.org/mediawiki/2017/3/35/T-Freiburg-7_19_2017_pH_change_3.png"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=no_acid_3.png" class="media" title="no_acid_3.png"><img src="/igem2017/lib/exe/fetch.php?w=200&amp;tok=2dd7e8&amp;media=no_acid_3.png" class="mediacenter" alt="" width="200" /></a></td>
 	</tr>
 	<tr class="row4">
-		<td class="col0"> 1µl </td><td class="col1"> T4 </td>
+		<td class="col0">2ml RPMI1640 + 65µl lactic acid (0.6 M)</td><td class="col1">6.2</td><td class="col2">95 min</td><td class="col3"><img style="max-height: 300px" src="https://static.igem.org/mediawiki/2017/d/de/T-Freiburg-7_19_2017_pH_change_4.png"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=07-20-17_lactic_acid_no_cells.png" class="media" title="07-20-17_lactic_acid_no_cells.png"><img src="/igem2017/lib/exe/fetch.php?w=200&amp;tok=8aeac1&amp;media=07-20-17_lactic_acid_no_cells.png" class="mediacenter" alt="" width="200" /></a></td>
 	</tr>
-</table></div>
+	<tr class="row5">
-<div class='secedit editbutton_table editbutton_235'> </div>
+		<td class="col0">2ml RPMI1649 + 65µl lactic acid (0.6 M)</td><td class="col1">6.3</td><td class="col2">90 min</td><td class="col3"><img style="max-height: 300px" src="https://static.igem.org/mediawiki/2017/8/8d/T-Freiburg-7_19_2017_pH_change_5.png"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=07-21-17_lactic_acid_no_cells.png" class="media" title="07-21-17_lactic_acid_no_cells.png"><img src="/igem2017/lib/exe/fetch.php?w=200&amp;tok=2ac7e7&amp;media=07-21-17_lactic_acid_no_cells.png" class="mediacenter" alt="" width="200" /></a></td>
-<p>
-Ligation was performed 37°C for 2 h.<br/>
-</p>
-</div>
-<div class='secedit editbutton_section editbutton_234'> </div>
-<h3 class="sectionedit236" id="digest_of_lenti-cas9_with_bsmbi3">Digest of lenti-Cas9 with BsmBI</h3>
-<div class="level3">
-<p>
-Digest reaction a)<br/>
-</p>
-<div class="table sectionedit237"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
 	</tr>
-	</thead>
+	<tr class="row6">
-	<tr class="row1">
+		<td class="col0">2ml RPMI1640 + 20µl HCl(1.2 M)</td><td class="col1">6.6</td><td class="col2">105 min</td><td class="col3"><img style="max-height: 300px" src="https://static.igem.org/mediawiki/2017/5/5f/T-Freiburg-7_19_2017_pH_change_6.png"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=07-20-17_hcl_no_cells.png" class="media" title="07-20-17_hcl_no_cells.png"><img src="/igem2017/lib/exe/fetch.php?w=200&amp;tok=3463ad&amp;media=07-20-17_hcl_no_cells.png" class="mediacenter" alt="" width="200" /></a></td>
-		<td class="col0">2.5 μl</td><td class="col1"> Plasmid (500ng/µl)</td>
 	</tr>
-	<tr class="row2">
+	<tr class="row7">
-		<td class="col0">1 µl </td><td class="col1"> BsmBI</td>
+		<td class="col0">2ml RPMI1640 + 30µl HCl(1.2 M)</td><td class="col1">6.6</td><td class="col2">100 min</td><td class="col3"><img style="max-height: 300px" src="https://static.igem.org/mediawiki/2017/c/cf/T-Freiburg-7_19_2017_pH_change_7.png"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=07-21-17_hcl_no_cells.png" class="media" title="07-21-17_hcl_no_cells.png"><img src="/igem2017/lib/exe/fetch.php?w=200&amp;tok=0e0744&amp;media=07-21-17_hcl_no_cells.png" class="mediacenter" alt="" width="200" /></a></td>
 	</tr>
-	<tr class="row3">
+	<tr class="row8">
-		<td class="col0">5 µl</td><td class="col1"> 3.1 10x buffer</td>
+		<td class="col0">7.5 ml RPMI1640 + 250.000 HEK cells / ml</td><td class="col1">7.3</td><td class="col2">24 h</td><td class="col3"><img style="max-height: 300px" src="https://static.igem.org/mediawiki/2017/4/44/T-Freiburg-7_19_2017_pH_change_8.png"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=250k_cells_no_acid.png" class="media" title="250k_cells_no_acid.png"><img src="/igem2017/lib/exe/fetch.php?w=200&amp;tok=9a20f6&amp;media=250k_cells_no_acid.png" class="mediacenter" alt="" width="200" /></a></td>
 	</tr>
-	<tr class="row4">
+	<tr class="row9">
-		<td class="col0">41.5 µl</td><td class="col1"> H2O</td>
+		<td class="col0">7.5 ml RPMI1640 + 500.000 HEK cells / ml</td><td class="col1">7.17</td><td class="col2">26 h</td><td class="col3"><img style="max-height: 300px" src="https://static.igem.org/mediawiki/2017/8/80/T-Freiburg-7_19_2017_pH_change_9.jpeg"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=ph-test_19-08-17.jpg" class="media" title="ph-test_19-08-17.jpg"><img src="/igem2017/lib/exe/fetch.php?w=200&amp;tok=0735d0&amp;media=ph-test_19-08-17.jpg" class="mediacenter" alt="" width="200" /></a></td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_237'> </div>
-<p>
-Digest reaction b)<br/>
-</p>
-<div class="table sectionedit238"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0">15 μl</td><td class="col1"> Plasmid (100ng/µl)</td>
-	</tr>
-	<tr class="row2">
-		<td class="col0">1 µl </td><td class="col1"> BsmBI</td>
-	</tr>
-	<tr class="row3">
-		<td class="col0">5 µl</td><td class="col1"> 3.1 10x buffer</td>
-	</tr>
-	<tr class="row4">
-		<td class="col0">29 µl</td><td class="col1"> H2O</td>
 	</tr>
 </table></div>
-<div class='secedit editbutton_table editbutton_238'> </div>
+<div class='secedit editbutton_table editbutton_54'> </div>
 </div>
-<div class='secedit editbutton_section editbutton_236'> </div>
+<div class='secedit editbutton_section editbutton_53'> </div>
-<h2 class="sectionedit239" id="section050717">05.07.17</h2>
+<h2 class="sectionedit55" id="section100817">10.08.17</h2>
 <div class="level2">
 </div>
-<div class='secedit editbutton_section editbutton_239'> </div>
+<div class='secedit editbutton_section editbutton_55'> </div>
-<h3 class="sectionedit240" id="colony_for_miniprep">Colony for miniprep</h3>
+<h3 class="sectionedit56" id="transformation_of_pwhe-644">Transformation of pWHE-644</h3>
 <div class="level3">
 <p>
-µl LB containing bacteria from colonies lenti-Cas9 c1 and c2 were incubated overnight.
+- transformation in DH5-α
 </p>
 </div>
-<div class='secedit editbutton_section editbutton_240'> </div>
+<div class='secedit editbutton_section editbutton_56'> </div>
-<h3 class="sectionedit241" id="digest_of_lenti-cas9_with_bsmbi4">Digest of lenti-Cas9 with BsmBI</h3>
+<h2 class="sectionedit57" id="section120817">12.08.17</h2>
-<div class="level3">
-<div class="table sectionedit242"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0">20 μl</td><td class="col1"> Plasmid (30ng/µl - 50 ng/µl)</td>
-	</tr>
-	<tr class="row2">
-		<td class="col0">1 µl </td><td class="col1"> BsmBI</td>
-	</tr>
-	<tr class="row3">
-		<td class="col0">5 µl</td><td class="col1"> 3.1 10x buffer</td>
-	</tr>
-	<tr class="row4">
-		<td class="col0">24 µl</td><td class="col1"> H2O</td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_242'> </div>
-</div>
-<div class='secedit editbutton_section editbutton_241'> </div>
-<h2 class="sectionedit243" id="section070717">07.07.17</h2>
 <div class="level2">
 </div>
-<div class='secedit editbutton_section editbutton_243'> </div>
+<div class='secedit editbutton_section editbutton_57'> </div>
-<h3 class="sectionedit244" id="digest_of_lenti-cas9_with_bsmbi5">Digest of lenti-Cas9 with BsmBI</h3>
+<h3 class="sectionedit58" id="miniprep_of_pwhe-644_in_dh3-α">Miniprep of pWHE-644 in DH3-α</h3>
 <div class="level3">
-<div class="table sectionedit245"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0">2 μl</td><td class="col1"> Plasmid (250 ng/µl, prepped the day before)</td>
-	</tr>
-	<tr class="row2">
-		<td class="col0">2 µl </td><td class="col1"> BsmBI</td>
-	</tr>
-	<tr class="row3">
-		<td class="col0">2.5 µl</td><td class="col1"> 3.1 10x buffer</td>
-	</tr>
-	<tr class="row4">
-		<td class="col0">18.5 µl</td><td class="col1"> H2O</td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_245'> </div>
 <p>
-Digest was performed at 55°C for 2 h.<br/>
+- miniprep with Zymo kit
 </p>
 </div>
-<div class='secedit editbutton_section editbutton_244'> </div>
+<div class='secedit editbutton_section editbutton_58'> </div>
-<h3 class="sectionedit246" id="dpni_digest_of_cas9-gfp_ligations">DpnI digest of Cas9-GFP ligations</h3>
+<h3 class="sectionedit59" id="test_digest_of_pwhe-644">Test digest of pWHE-644</h3>
 <div class="level3">
 <p>
-sgRNA1 (PCR on 30.05.17, ligation on 01.06.17)<br/>
+µl NcoI<br/>
-sgRNA4 (PCR on 10.06.17, ligation on 13.06.17)<br/>
+µl DNA<br/>
-sgRNA9 (PCR on 10.06.17, ligation on 13.06.17)<br/>
+.5µl FD-buffer 10x<br/>
-sgRNA20 (PCR on 14.06.17, ligation on 16.06.17)<br/>
+.5µl H20<br/>
-</p>
+Excepted bands could be observed.<br/>
-<p>
-Digest reaction a) for each sgRNA
 </p>
-<div class="table sectionedit247"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0">1 μl</td><td class="col1"> DpnI</td>
-	</tr>
-	<tr class="row2">
-		<td class="col0">1 µl </td><td class="col1"> CutSmart</td>
-	</tr>
-	<tr class="row3">
-		<td class="col0">2 µl</td><td class="col1"> ligation </td>
-	</tr>
-	<tr class="row4">
-		<td class="col0">6 µl</td><td class="col1"> H2O</td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_247'> </div>
-<p>
-Digest reaction b) for each sgRNA
-</p>
-<div class="table sectionedit248"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0">1 μl</td><td class="col1"> DpnI</td>
-	</tr>
-	<tr class="row2">
-		<td class="col0">1 µl </td><td class="col1"> CutSmart</td>
-	</tr>
-	<tr class="row3">
-		<td class="col0">4 µl</td><td class="col1"> ligation </td>
-	</tr>
-	<tr class="row4">
-		<td class="col0">4 µl</td><td class="col1"> H2O</td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_248'> </div>
-<p>
-Transformation into XL10gold performed according to heat shock protocol (06.05.17).<br/>
-</p>
+</div>
+<div class='secedit editbutton_section editbutton_59'> </div>
+<h2 class="sectionedit60" id="section130817">13.08.17</h2>
+<div class="level2">
 </div>
-<div class='secedit editbutton_section editbutton_246'> </div>
+<div class='secedit editbutton_section editbutton_60'> </div>
-<h3 class="sectionedit249" id="test_digest_of_cas9-gfp_sgrna1_and_sgrna9">Test digest of Cas9-GFP sgRNA1 and sgRNA9</h3>
+<h3 class="sectionedit61" id="miniprep_of_cmv-vegfr2_pig_138">Miniprep of CMV-VEGFR2 (pIG_138)</h3>
 <div class="level3">
 <p>
-Cas9-GFP sgRNA1 colonies (PCR on 30.05.17)<br/>
+- 7x miniprep of CMV-VEGFR2
 </p>
-<div class="table sectionedit250"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0">0.5 μl</td><td class="col1"> NdeI</td>
-	</tr>
-	<tr class="row2">
-		<td class="col0">x µl </td><td class="col1"> Cas0-GFP sgRNA1 plasmid (1µg)</td>
-	</tr>
-	<tr class="row3">
-		<td class="col0">1 µl</td><td class="col1"> CutSmart bufffer </td>
-	</tr>
-	<tr class="row4">
-		<td class="col0">8.5-x µl</td><td class="col1"> H2O</td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_250'> </div>
-<p>
-Cas9-GFP sgRNA9 colonies (PCR on 10.06.17)<br/>
-</p>
-<div class="table sectionedit251"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0">0.25 μl</td><td class="col1"> NcoI</td>
-	</tr>
-	<tr class="row2">
-		<td class="col0">x µl </td><td class="col1"> Cas0-GFP sgRNA1 plasmid (1µg)</td>
-	</tr>
-	<tr class="row3">
-		<td class="col0">1 µl</td><td class="col1"> CutSmart bufffer </td>
-	</tr>
-	<tr class="row4">
-		<td class="col0">8.75-x µl</td><td class="col1"> H2O</td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_251'> </div>
 </div>
-<div class='secedit editbutton_section editbutton_249'> </div>
+<div class='secedit editbutton_section editbutton_61'> </div>
-<h3 class="sectionedit252" id="heatshock_transformation_of_cas9-gfp_plasmids">Heatshock transformation of Cas9-GFP plasmids</h3>
+<h3 class="sectionedit62" id="test_digest_of_cmv-vegfr2_pig_138">Test digest of CMV-VEGFR2 (pIG_138)</h3>
 <div class="level3">
 <p>
-Transformation of Cas9-GFP sgRNA3 colonies 3/6 and sgRNAscramble colonies 2/4 into XL10gold was performed according to heatshock protocol.<br/>
+µl DNA<br/>
-</p>
+.5µl buffer<br/>
-</div>
+µl BamHI<br/>
-<div class='secedit editbutton_section editbutton_252'> </div>
-<h2 class="sectionedit253" id="section080717">08.07.17</h2>
-<div class="level2">
-</div>
+.5µl H20<br/>
-<div class='secedit editbutton_section editbutton_253'> </div>
-<h3 class="sectionedit254" id="test_digest_of_sgrna3_and_sgrna_scramble">Test digest of sgRNA3 and sgRNA scramble</h3>
-<div class="level3">
-<div class="table sectionedit255"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">a</th><th class="col1">b</th><th class="col2">c</th><th class="col3">d</th><th class="col4">component</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0"> 1 µl </td><td class="col1"> 1 µl </td><td class="col2"> 2 µl </td><td class="col3"> 2 µl </td><td class="col4"> NdeI </td>
-	</tr>
-	<tr class="row2">
-		<td class="col0"> 4 µl </td><td class="col1"> - </td><td class="col2"> 4 µl </td><td class="col3"> - </td><td class="col4"> sgRNA c4 (250 ng/µl)</td>
-	</tr>
-	<tr class="row3">
-		<td class="col0"> - </td><td class="col1"> 8 µl </td><td class="col2"> - </td><td class="col3"> 8 µl </td><td class="col4"> scramble c3 (250 ng/µl)</td>
-	</tr>
-	<tr class="row4">
-		<td class="col0"> 1 µl </td><td class="col1"> 1 µl </td><td class="col2"> 1 µl </td><td class="col3"> 1 µl </td><td class="col4"> CutSmart </td>
-	</tr>
-	<tr class="row5">
-		<td class="col0"> 4 µl </td><td class="col1"> - </td><td class="col2"> 4 µl </td><td class="col3"> - </td><td class="col4"> H2O </td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_255'> </div>
-<p>
-Digest was performed at 37°C for 2 h.<br/>
-</p>
+Only one of the expected bands could be oberserved.<br/>
-</div>
-<div class='secedit editbutton_section editbutton_254'> </div>
-<h3 class="sectionedit256" id="colonies_picked">Colonies picked</h3>
-<div class="level3">
-<p>
-Colonies were picked for sgRNA4 and sgRNA1 (5 colonies each) and sgRNA9 (2 colonies).
-Since no colony grew for sgRNA 20, the plate was incubated a second night. One picked for each re-trafo.
 </p>
 </div>
-<div class='secedit editbutton_section editbutton_256'> </div>
+<div class='secedit editbutton_section editbutton_62'> </div>
-<h2 class="sectionedit257" id="section090717">09.07.17</h2>
+<h2 class="sectionedit63" id="section1308171">13.08.17</h2>
 <div class="level2">
 </div>
-<div class='secedit editbutton_section editbutton_257'> </div>
+<div class='secedit editbutton_section editbutton_63'> </div>
-<h3 class="sectionedit258" id="test_digest_of_5_colonies_sgrna1_in_cas9-gfp">Test digest of 5 colonies sgRNA1 in Cas9-GFP</h3>
+<h3 class="sectionedit64" id="test_digest_of_cmv-vegfr2_pig_1381">Test digest of CMV-VEGFR2 (pIG_138)</h3>
 <div class="level3">
-<div class="table sectionedit259"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0"> x µl </td><td class="col1"> plasmid (1 µg) </td>
-	</tr>
-	<tr class="row2">
-		<td class="col0"> 1 µl </td><td class="col1"> NdeI </td>
-	</tr>
-	<tr class="row3">
-		<td class="col0"> 1 µl </td><td class="col1"> CutSmart </td>
-	</tr>
-	<tr class="row4">
-		<td class="col0"> 8-x µl </td><td class="col1"> H2O </td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_259'> </div>
 <p>
-Digest was performed at 37°C for 2 h.<br/>
+µl DNA<br/>
-sgRNA scramble, colony 3 negative control, colony 4 positive control.<br/>
+.5µl FD-buffer<br/>
-</p>
+µl SmaI<br/>
-</div>
+Only one of the expected bands could be oberserved.<br/>
-<div class='secedit editbutton_section editbutton_258'> </div>
-<h3 class="sectionedit260" id="re-trafos_sgrna3_scramble">Re-trafos sgRNA3/scramble</h3>
-<div class="level3">
-<p>
-From overnight cultures of sgRNA3 and sgRNAscramble re-trafos 2 glycerol stocks, 1 miniprep (5.5 ml) and 1 overnight culture for midiprep (100ml made with 4 ml cultured bacter9a) were made for each sgRNA.<br/>
-From overnight cultures of sgRNA1, sgRNA4 and sgRNA9 re-trafos 4 glycerol stocks and 4 minipreps were made for all sgRNAs.
 </p>
 </div>
-<div class='secedit editbutton_section editbutton_260'> </div>
+<div class='secedit editbutton_section editbutton_64'> </div>
-<h2 class="sectionedit261" id="section100717">10.07.17</h2>
+<h2 class="sectionedit65" id="section160817">16.08.17</h2>
 <div class="level2">
 </div>
-<div class='secedit editbutton_section editbutton_261'> </div>
+<div class='secedit editbutton_section editbutton_65'> </div>
-<h3 class="sectionedit262" id="restriction_digest_of_knockdown_backbone_sden">Restriction digest of knockdown backbone SDEN</h3>
+<h3 class="sectionedit66" id="ph_test_with_jurkat_cells">pH test with Jurkat cells</h3>
 <div class="level3">
-<div class="table sectionedit263"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0"> 6.3 µl </td><td class="col1 rightalign">  SGEN backbone (5 µg) </td>
-	</tr>
-	<tr class="row2">
-		<td class="col0"> 2 µl </td><td class="col1"> 10x CutSmart </td>
-	</tr>
-	<tr class="row3">
-		<td class="col0"> 1 µl </td><td class="col1"> EcoRI </td>
-	</tr>
-	<tr class="row4">
-		<td class="col0"> 1 µl </td><td class="col1"> XhoI </td>
-	</tr>
-	<tr class="row5">
-		<td class="col0"> 9.7 µl </td><td class="col1"> H2O </td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_263'> </div>
-<p>
-Digest was performed for 2 h at 37°C.<br/>
-Inactivation took 20 min at 65°C.<br/>
-</p>
 <p>
-Dephosphorylation<br/>
+- centrifuge Jurkatt cells, dilute in RPMI1640<br/>
-</p>
+- count cells<br/>
-<div class="table sectionedit264"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0"> 10 µl </td><td class="col1"> antarctic phosphatase buffer </td>
-	</tr>
-	<tr class="row2">
-		<td class="col0"> 1 µl </td><td class="col1"> antarctic phosphatase </td>
-	</tr>
-	<tr class="row3">
-		<td class="col0"> 10 µl </td><td class="col1"> digested plasmid </td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_264'> </div>
-<p>
-Dephosphorylation took 10 min at 37°C.<br/>
-</p>
+- incubation of Jurkat cells and lactic acid<br/>
-</div>
+- pH measurements after 0h, 30min, 1h and 1h30min<br/>
-<div class='secedit editbutton_section editbutton_262'> </div>
-<h3 class="sectionedit265" id="restriction_digest_of_pcr_fragments_of_knockdown_inserts">Restriction digest of PCR fragments of knockdown inserts</h3>
-<div class="level3">
-<div class="table sectionedit266"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">GelEx (22.06.17)</th><th class="col1">PCREx (27.06.17)</th><th class="col2">component</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0"> 9.1 ng/µl </td><td class="col1"> 11.0 ng/µl </td><td class="col2"> olG17_112 </td>
-	</tr>
-	<tr class="row2">
-		<td class="col0"> 4.5 ng/µl </td><td class="col1"> 5.7 ng/µl </td><td class="col2"> olG17_113 </td>
-	</tr>
-	<tr class="row3">
-		<td class="col0"> 8.9 ng/µl </td><td class="col1"> 5.6 ng/µl </td><td class="col2"> olG17_114 </td>
-	</tr>
-	<tr class="row4">
-		<td class="col0"> 11.3 ng/µl </td><td class="col1"> 7.9 ng/µl </td><td class="col2"> olG17_115 </td>
-	</tr>
-	<tr class="row5">
-		<td class="col0"> 5.9 ng/µl </td><td class="col1"> 6.3 ng/µl </td><td class="col2"> olG17_116 </td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_266'> </div>
-<p>
-All 10 samples were digested completely.
-</p>
-<div class="table sectionedit267"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0"> 25 µl </td><td class="col1"> PCR-Fragments</td>
-	</tr>
-	<tr class="row2">
-		<td class="col0">0.5 µl </td><td class="col1"> 10x CutSmart</td>
-	</tr>
-	<tr class="row3">
-		<td class="col0"> 0.25 µl </td><td class="col1"> XhoI </td>
-	</tr>
-	<tr class="row4">
-		<td class="col0"> 0.25 µl </td><td class="col1"> EcoRI </td>
-	</tr>
-	<tr class="row5">
-		<td class="col0"> 19.5 µl </td><td class="col1"> H2O</td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_267'> </div>
-<p>
-Digest was performed for 3 h at 37°C.<br/>
-Inactivation took 20 min at 65°C.<br/>
+- 1) only RPMI1640 with cells, start pH at 7.2<br/>
-Fragments were separated using gel electrophoresis (2% agarose, 120 V, 30 min).<br/>
+- 2) RPMI1640 with cells and 100µl lactic acid, start pH at 6.8<br/>
-The digested fragments were not visible in the gel, the phosphorylated backbone smeared (Picture: IM.000500.Tif). Only the non-dephosphorylated was backbone cut out and extracted from gel using a manufacturer&#039;s protocol. <br/>
 </p>
-</div>
-<div class='secedit editbutton_section editbutton_265'> </div>
-<h2 class="sectionedit268" id="midiprep_cas9-gfp_sgrna3_c6_scramble_c4">Midiprep Cas9-GFP sgRNA3(c6)/scramble(c4)</h2>
-<div class="level2">
 <p>
-Midiprep was performed according to manufacturer&#039;s protocol to obtain plasmids for electroporation from sequenced colonies (150 µg).
+after 0h:<br/>
-</p>
-</div>
-<div class='secedit editbutton_section editbutton_268'> </div>
-<h3 class="sectionedit269" id="site-directed_mutagenesis_pcr_cas9-gfp_sgrna3_sgrna20">Site-directed mutagenesis PCR CAS9-GFP sgRNA3 &amp; sgRNA20</h3>
-<div class="level3">
-<p>
-PCR was performed with Q5 Master Mix for sgRNA3 and self-mixed solution for sgRNA 20.
 </p>
+<div class="table sectionedit67"><table class="inline">
-</div>
-<div class='secedit editbutton_section editbutton_269'> </div>
-<h2 class="sectionedit270" id="section110717">11.07.17</h2>
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_270'> </div>
-<h3 class="sectionedit271" id="pcr_of_97meres_for_knockdown_inserts">PCR of 97meres for knockdown inserts</h3>
-<div class="level3">
-<div class="table sectionedit272"><table class="inline">
 	<thead>
 	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
+		<th class="col0">cell concentration (cells/ml)</th><th class="col1">pH of 1)</th><th class="col2">pH of 2)</th>
 	</tr>
 	</thead>
 	<tr class="row1">
-		<td class="col0"> 10 µl </td><td class="col1"> 5x Q5 buffer </td>
+		<td class="col0">3mio</td><td class="col1">7.46</td><td class="col2">6.81</td>
 	</tr>
 	<tr class="row2">
-		<td class="col0"> 1 µl </td><td class="col1"> dNTPs (10 mM) </td>
+		<td class="col0">2mio</td><td class="col1">7.41</td><td class="col2">6.82</td>
 	</tr>
 	<tr class="row3">
-		<td class="col0"> 2.5 µl </td><td class="col1"> 5&#039;mirEXhoIfwd (10 µM) </td>
+		<td class="col0">1.5mio</td><td class="col1">7.47</td><td class="col2">6.86</td>
 	</tr>
 	<tr class="row4">
-		<td class="col0"> 2.5 µl </td><td class="col1"> 3&#039;mirEEcoRIrev (10 µM) </td>
+		<td class="col0">1mio</td><td class="col1">7.49</td><td class="col2">6.79</td>
 	</tr>
 	<tr class="row5">
-		<td class="col0"> 2 µl </td><td class="col1"> 97mere template (0.01 ng/µl) </td>
+		<td class="col0">500k</td><td class="col1">7.45</td><td class="col2">6.78</td>
 	</tr>
 	<tr class="row6">
-		<td class="col0"> 0.5 µl </td><td class="col1"> Q5 Pol </td>
+		<td class="col0">100k</td><td class="col1">7.45</td><td class="col2">6.77</td>
-	</tr>
-	<tr class="row7">
-		<td class="col0"> 31.5 µl </td><td class="col1"> H2O </td>
 	</tr>
 </table></div>
-<div class='secedit editbutton_table editbutton_272'> </div>
+<div class='secedit editbutton_table editbutton_67'> </div>
 <p>
-Templates: 0lG17_112 to olG17_116<br/>
+after 30min:<br/>
 </p>
-<div class="table sectionedit273"><table class="inline">
+<div class="table sectionedit68"><table class="inline">
 	<thead>
 	<tr class="row0">
-		<th class="col0">step</th><th class="col1">temperature</th><th class="col2">duration</th>
+		<th class="col0">cell concentration (cells/ml)</th><th class="col1">pH of 1)</th><th class="col2">pH of 2)</th>
 	</tr>
 	</thead>
 	<tr class="row1">
-		<td class="col0"> 1 </td><td class="col1"> 98°C </td><td class="col2"> 1 min </td>
+		<td class="col0">3mio</td><td class="col1">7.52</td><td class="col2">7.18</td>
 	</tr>
 	<tr class="row2">
-		<td class="col0"> 2 </td><td class="col1"> 98°C </td><td class="col2"> 10 sec </td>
+		<td class="col0">2mio</td><td class="col1">7.51</td><td class="col2">7.13</td>
 	</tr>
 	<tr class="row3">
-		<td class="col0"> 3 </td><td class="col1"> 66°C </td><td class="col2"> 30 sec </td>
+		<td class="col0">1.5mio</td><td class="col1">7.53</td><td class="col2">7.14</td>
 	</tr>
 	<tr class="row4">
-		<td class="col0"> 4 </td><td class="col1"> 72°C </td><td class="col2"> 20 sec </td>
+		<td class="col0">1mio</td><td class="col1">7.52</td><td class="col2">7.11</td>
 	</tr>
 	<tr class="row5">
-		<td class="col0"> 5 </td><td class="col1"> 72°C </td><td class="col2"> 2 min </td>
+		<td class="col0">500k</td><td class="col1">7.54</td><td class="col2">7.07</td>
 	</tr>
 	<tr class="row6">
-		<td class="col0"> 6 </td><td class="col1"> 8°C </td><td class="col2 leftalign">  </td>
+		<td class="col0">100k</td><td class="col1">7.55</td><td class="col2">7.12</td>
 	</tr>
 </table></div>
-<div class='secedit editbutton_table editbutton_273'> </div>
+<div class='secedit editbutton_table editbutton_68'> </div>
 <p>
-Steps 2 to 4 were repeated 33x.<br/>
+after 1h:<br/>
-Expected product of 144 bp was observed (picture no. IM000506.Tif) and extracted from gel with Qiagen PCR extraction kit according to manufacturer&#039;s protocol and eluted in 30 µl H2O.<br/>
 </p>
-<div class="table sectionedit274"><table class="inline">
+<div class="table sectionedit69"><table class="inline">
 	<thead>
 	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
+		<th class="col0">cell concentration (cells/ml)</th><th class="col1">pH of 1)</th><th class="col2">pH of 2)</th>
 	</tr>
 	</thead>
 	<tr class="row1">
-		<td class="col0"> 5 µl </td><td class="col1"> 10x CutSmart buffer </td>
+		<td class="col0">3mio</td><td class="col1">7.48</td><td class="col2">7.26</td>
 	</tr>
 	<tr class="row2">
-		<td class="col0"> 0.25 µl </td><td class="col1"> XhoI </td>
+		<td class="col0">2mio</td><td class="col1">7.57</td><td class="col2">7.22</td>
 	</tr>
 	<tr class="row3">
-		<td class="col0"> 0.25 µl </td><td class="col1"> EcoRI </td>
+		<td class="col0">1.5mio</td><td class="col1">7.60</td><td class="col2">7.24</td>
 	</tr>
 	<tr class="row4">
-		<td class="col0"> 4.5 µl </td><td class="col1"> H2O </td>
+		<td class="col0">1mio</td><td class="col1">7.60</td><td class="col2">7.22</td>
 	</tr>
 	<tr class="row5">
-		<td class="col0"> 40 µl </td><td class="col1"> template </td>
+		<td class="col0">500k</td><td class="col1">7.60</td><td class="col2">7.18</td>
+	</tr>
+	<tr class="row6">
+		<td class="col0">100k</td><td class="col1">7.63</td><td class="col2">7.21</td>
 	</tr>
 </table></div>
-<div class='secedit editbutton_table editbutton_274'> </div>
+<div class='secedit editbutton_table editbutton_69'> </div>
 <p>
-Previously eluted product was digested for 3 h at 37°C (10 µ from each PCR run were mixed, for each of the 5 templates).
+after 1h30min:<br/>
-Fragments were separated using gel electrophoresis (2% agarose gel, 120 V, 20 min) but gel was empty apart from ladder (picture no. IM000504.Tif).<br/>
 </p>
+<div class="table sectionedit70"><table class="inline">
-<p>
-Troubleshooting showed that PCR extraction had not been successful (gel 120 V, 20 min, picture IM000510.Tif).<br/>
-</p>
-</div>
-<div class='secedit editbutton_section editbutton_271'> </div>
-<h3 class="sectionedit275" id="digest_sgen_backbone_of_gibson_colonies_7_and_2">Digest SGEN backbone of Gibson colonies 7 and 2</h3>
-<div class="level3">
-<div class="table sectionedit276"><table class="inline">
 	<thead>
 	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
+		<th class="col0">cell concentration (cells/ml)</th><th class="col1">pH of 1)</th><th class="col2">pH of 2)</th>
 	</tr>
 	</thead>
 	<tr class="row1">
-		<td class="col0"> 6.3 µl </td><td class="col1"> SGEN plasmid (5 µg) </td>
+		<td class="col0">3mio</td><td class="col1">7.42</td><td class="col2">7.14</td>
 	</tr>
 	<tr class="row2">
-		<td class="col0"> 2 µl </td><td class="col1"> CutSmart buffer </td>
+		<td class="col0">2mio</td><td class="col1">7.55</td><td class="col2">7.27</td>
 	</tr>
 	<tr class="row3">
-		<td class="col0"> 1 µl </td><td class="col1"> EcoRI </td>
+		<td class="col0">1.5mio</td><td class="col1">7.46</td><td class="col2">7.35</td>
 	</tr>
 	<tr class="row4">
-		<td class="col0"> 1 µl </td><td class="col1"> XhoI </td>
+		<td class="col0">1mio</td><td class="col1">7.62</td><td class="col2">7.18</td>
 	</tr>
 	<tr class="row5">
-		<td class="col0"> 9.7 µl </td><td class="col1"> H2O </td>
+		<td class="col0">500k</td><td class="col1">7.51</td><td class="col2">7.23</td>
+	</tr>
+	<tr class="row6">
+		<td class="col0">100k</td><td class="col1">7.59</td><td class="col2">7.26</td>
 	</tr>
 </table></div>
-<div class='secedit editbutton_table editbutton_276'> </div>
+<div class='secedit editbutton_table editbutton_70'> </div>
-<p>
-Digest was performed for 2 h at 37 °C.<br/>
-Fragments were separated using gel electrophoresis (picture no. IM000502.Tif) and extracted with qiagen gel extraction kit according to manufacturer&#039;s protocol.
-</p>
 </div>
-<div class='secedit editbutton_section editbutton_275'> </div>
+<div class='secedit editbutton_section editbutton_66'> </div>
-<h2 class="sectionedit277" id="section120717">12.07.17</h2>
+<h2 class="sectionedit71" id="section1608171">16.08.17</h2>
 <div class="level2">
 </div>
-<div class='secedit editbutton_section editbutton_277'> </div>
+<div class='secedit editbutton_section editbutton_71'> </div>
-<h3 class="sectionedit278" id="overnight_cultures_of_ko-kit_plasmids_and_lenti-cas9">Overnight cultures of KO-Kit plasmids and lenti-Cas9</h3>
+<h3 class="sectionedit72" id="test_digest_of_cmv-vegfr2_pig_1382">Test digest of CMV-VEGFR2 (pIG_138)</h3>
 <div class="level3">
 <p>
-Overnight cultures of KO-Kit 101, 104, 203, lenti-Cas9 for minipreps and midipreps were prepared (13.07./14.07.).
+µl DNA<br/>
-</p>
-</div>
+.5µl FD-buffer<br/>
-<div class='secedit editbutton_section editbutton_278'> </div>
-<h3 class="sectionedit279" id="gels_for_checking_purified_knockdown_insert_dna">Gels for checking purified knockdown insert DNA</h3>
-<div class="level3">
-<p>
+µl BamHI<br/>
-No DNA was visible on gel since concentrations were too low (pictures no. IM000512.Tif and IM000514.Tif).<br/>
+,5µl H20<br/>
 </p>
 </div>
-<div class='secedit editbutton_section editbutton_279'> </div>
+<div class='secedit editbutton_section editbutton_72'> </div>
-<h3 class="sectionedit280" id="purification_restriction_digest_and_ligation_of_knockdown_insert_pcr_products">Purification, restriction digest and ligation of knockdown insert PCR products</h3>
+<h3 class="sectionedit73" id="ctla4_induction">Ctla4 induction</h3>
 <div class="level3">
 <p>
-Purification of PCR products of all 5 PCRs (picture IM000520.Tif) was performed using Qiagen gel extraction kit according to manufacturer&#039;s protocol. Samples washed twice with PE buffer show better curve on nanodrop.<br/>
+- Jurkat cells transiently transfected with Ctla4-GFP-CMV-mCherry<br/>
-</p>
+- positive control: cotransfected with CMV-GFP and CMV mCherry<br/>
-<p>
+- add VEGF<br/>
-Restriction digest of purified samples (samples from gel using 20x blue loading dye)<br/>
-</p>
+- incubation for 24h at 37°C<br/>
-<div class="table sectionedit281"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0"> 17 µl </td><td class="col1"> purified DNA </td>
-	</tr>
-	<tr class="row2">
-		<td class="col0"> 2 µl </td><td class="col1"> 10x CutSmart buffer </td>
-	</tr>
-	<tr class="row3">
-		<td class="col0"> 0.5 µl </td><td class="col1"> EcoRI </td>
-	</tr>
-	<tr class="row4">
-		<td class="col0"> 0.5 µl </td><td class="col1"> XhoI </td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_281'> </div>
-<p>
-h at 37°C<br/>
-Fragments were purified using Qiagen purification kit according to maufacturer&#039;s protocol.
-</p>
-<p>
-ligation<br/>
-</p>
-<div class="table sectionedit282"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0"> 0.7 µl </td><td class="col1"> digested backbone (50 ng) </td>
-	</tr>
-	<tr class="row2">
-		<td class="col0"> x µl </td><td class="col1"> digested insert (2,415 ng) </td>
-	</tr>
-	<tr class="row3">
-		<td class="col0"> 1 µl </td><td class="col1"> T4 ligase buffer </td>
-	</tr>
-	<tr class="row4">
-		<td class="col0"> 1 µl </td><td class="col1"> T4 ligase </td>
-	</tr>
-	<tr class="row5">
-		<td class="col0"> 7.3-x µ </td><td class="col1"> H2O </td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_282'> </div>
-</div>
-<div class='secedit editbutton_section editbutton_280'> </div>
-<h3 class="sectionedit283" id="gel_extraction_of_sgrna3_inserted_by_sdm_pcr_into_cas9-gfp">Gel extraction of sgRNA3 inserted by SDM PCR into CAS9-GFP</h3>
-<div class="level3">
-<p>
-Site-directed mutagenesis PCR of sgRNA20 into Cas9-GFP was repeated and failed.<br/>
-overnight cultures of Cas9-GFP-sgRNA5 were prepared in order to obtain DNA for electroporation. Electrophoresis on agarose of miniprepped coloniees confirm that CutSmart was contaminated with DNAse but not the DNA samples (clear bands were observed as expected).
 </p>
 </div>
-<div class='secedit editbutton_section editbutton_283'> </div>
+<div class='secedit editbutton_section editbutton_73'> </div>
-<h2 class="sectionedit284" id="section130717">13.07.17</h2>
+<h2 class="sectionedit74" id="section170817">17.08.17</h2>
 <div class="level2">
 </div>
-<div class='secedit editbutton_section editbutton_284'> </div>
+<div class='secedit editbutton_section editbutton_74'> </div>
-<h3 class="sectionedit285" id="sequencing_sgrnas_in_cas9-gfp">Sequencing sgRNAs in Cas9-GFP</h3>
+<h3 class="sectionedit75" id="pei_transfection_of_hek_cells">PEI transfection of HEK cells</h3>
 <div class="level3">
 <p>
-DNA from colonies of original trafo with CAS9-GFP sgRNAs 1, 4 and 9, which were not yet sequenced or where sequencing hat previously failed, were send to sequencing.<br/>
+- CMV-GFP from iGEM<br/>
+- CMV-GFP from tool box<br/>
-Minipreps(2x) of sgRNA5 in Cas9-GFP were performed according to manufacturer&#039;s protocol for 2 electroporations
 </p>
 </div>
-<div class='secedit editbutton_section editbutton_285'> </div>
+<div class='secedit editbutton_section editbutton_75'> </div>
-<h3 class="sectionedit286" id="heat_shock_with_ligated_knockdown_plasmids">Heat shock with ligated knockdown plasmids</h3>
+<h3 class="sectionedit76" id="flow_cytometry">Flow cytometry</h3>
 <div class="level3">
 <p>
-XL10gold bacteria were transformed with ligation sample (5 µl) following the heat shock protocol (5 plates containing plasmids based on olG17_112, olG17_113, olG17_114, olG17_115, olG17_116) Heat shock failed (bacteria were previously stored overnight at -20°C instead of -80°C, only 40 µl bacteria solution available per heat shock).
+- Jurkat with Ctla4-GFP-CMV-mCherry from 16.08.17
 </p>
+<div class="table sectionedit77"><table class="inline">
+	<thead>
+	<tr class="row0">
+		<th class="col0">mCherry</th><th class="col1">GFP (measured in green fluorescence channel)</th>
+	</tr>
+	</thead>
+	<tr class="row1">
+		<td class="col0"><img style="max-height: 500px" src="https://static.igem.org/mediawiki/2017/b/ba/T-Freiburg-8_17_2017_flow_cytometry_1.png"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=facs_mcherry_17-08-17.png" class="media" title="facs_mcherry_17-08-17.png"><img src="/igem2017/lib/exe/fetch.php?w=200&amp;tok=4ac1ce&amp;media=facs_mcherry_17-08-17.png" class="mediacenter" alt="" width="200" /></a></td><td class="col1"><img style="max-height: 500px" src="https://static.igem.org/mediawiki/2017/9/99/T-Freiburg-8_17_2017_flow_cytometry_2.png"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=facs_gfp_17-08-17.png" class="media" title="facs_gfp_17-08-17.png"><img src="/igem2017/lib/exe/fetch.php?w=200&amp;tok=3b20df&amp;media=facs_gfp_17-08-17.png" class="mediacenter" alt="" width="200" /></a></td>
+	</tr>
+</table></div>
+<div class='secedit editbutton_table editbutton_77'> </div>
 </div>
-<div class='secedit editbutton_section editbutton_286'> </div>
+<div class='secedit editbutton_section editbutton_76'> </div>
-<h2 class="sectionedit287" id="section150717">15.07.17</h2>
+<h2 class="sectionedit78" id="section180817">18.08.17</h2>
 <div class="level2">
 </div>
-<div class='secedit editbutton_section editbutton_287'> </div>
+<div class='secedit editbutton_section editbutton_78'> </div>
-<h3 class="sectionedit288" id="overnight_culture_of_xl10gold">Overnight culture of XL10gold</h3>
+<h3 class="sectionedit79" id="biorad_electroporation">BioRad electroporation</h3>
 <div class="level3">
 <p>
-An overnight culture of XL10 gold was prepared using 4 ml LB.
+Cell lines: Jurkat<br/>
+Plasmids: pIG_037, pIG_031, contransfection CMV-GFP<br/>
 </p>
+<ol>
+<li class="level1"><div class="li"> Count the cells</div>
+</li>
+<li class="level1"><div class="li"> Transfer 1 mio. cells into falcons and centrifuge with 100g for 3 min</div>
+</li>
+<li class="level1"><div class="li"> Resuspend the cell pellet with 100 µl P3 Buffer</div>
+</li>
+<li class="level1"><div class="li"> Add 9 µg pIG17_009</div>
+</li>
+<li class="level1"><div class="li"> Transfer the mixture into cuvette </div>
+</li>
+<li class="level1"><div class="li"> BioRad setting: 250 V, 960 µF, 35 sec.</div>
+</li>
+<li class="level1"><div class="li"> Add 500 µl RPMI medium to the cells immediately after electroporation</div>
+</li>
+<li class="level1"><div class="li"> Plate the cells into 12-well plate with 2 ml Medium</div>
+</li>
+<li class="level1"><div class="li"> Over night culturing</div>
+</li>
+<li class="level1"><div class="li"> Wash the cuvettes for reusing</div>
+</li>
+</ol>
 </div>
-<div class='secedit editbutton_section editbutton_288'> </div>
+<div class='secedit editbutton_section editbutton_78'> </div>
-<h2 class="sectionedit289" id="section160717">16.07.17</h2>
+<h3 class="sectionedit79" id="calcium_test">Calcium test</h3>
-   <img src="       https://static.igem.org/mediawiki/2017/6/68/T%E2%80%93FREIBURG%E2%80%93KO315.png" width="500px">
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_289'> </div>
-<h3 class="sectionedit290" id="overnight_culture_of_xl10gold1">overnight culture of XL10gold</h3>
 <div class="level3">
 <p>
-Overnight culture of XL10gold was enlagred to 50 ml LB.<br/>
+- incubation of cells at 37°C in FACS tubes for 5min
+- pre-FACS after 3min
-ZymoBroth start: 20:00-9:30
+- add VEGF (30ng/ml, 70ng/ml)
+- FACS afer 7min: no signal
 </p>
 </div>
-<div class='secedit editbutton_section editbutton_290'> </div>
+<div class='secedit editbutton_section editbutton_79'> </div>
-<h2 class="sectionedit291" id="section170717">17.07.17</h2>
+<h2 class="sectionedit80" id="section200817">20.08.17</h2>
 <div class="level2">
 </div>
-<div class='secedit editbutton_section editbutton_291'> </div>
+<div class='secedit editbutton_section editbutton_80'> </div>
-<h3 class="sectionedit292" id="culturing_xl10gold">Culturing XL10gold</h3>
+<h3 class="sectionedit81" id="biorad_electroporation1">Biorad electroporation</h3>
 <div class="level3">
 <p>
-Start value OD600 = 0.079<br/>
+Cell lines: Jurkat<br/>
-Using 1:10 dilution –&gt; OD600 = 0.79
+Plasmids: pIG_034, cotransfection CMV-GFP and CMV-mCherry, negative control (no DNA)<br/>
-</p>
-</div>
-<div class='secedit editbutton_section editbutton_292'> </div>
-<h3 class="sectionedit293" id="digest_cas9-gfp">Digest Cas9-GFP</h3>
-<div class="level3">
-<div class="table sectionedit294"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">components</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0"> 0.5 µl </td><td class="col1"> Cas9-GFP (1.1 µg/µl, 11.06.17) </td>
-	</tr>
-	<tr class="row2">
-		<td class="col0"> 0.25 µl </td><td class="col1"> EcoRI </td>
-	</tr>
-	<tr class="row3">
-		<td class="col0"> 0.25 µl </td><td class="col1"> NotI </td>
-	</tr>
-	<tr class="row4">
-		<td class="col0"> 1 µl </td><td class="col1"> CutSmart </td>
-	</tr>
-	<tr class="row5">
-		<td class="col0"> 8 µl </td><td class="col1"> H2O </td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_294'> </div>
-<p>
-Expected fragments: 3300 bp, 60000 bp (picture no. 529)<br/>
-Fragments were observed and 3.3 kb fragment was excised for extraction.
 </p>
+<ol>
+<li class="level1"><div class="li"> Count the cells<br/>
 </div>
-<div class='secedit editbutton_section editbutton_293'> </div>
+</li>
-<h3 class="sectionedit295" id="heatshock_transformation_of_knockdown_ligations_and_lenti-cas9">Heatshock transformation of knockdown ligations and lenti-CAS9</h3>
+<li class="level1"><div class="li"> Transfer 1 mio. cells into falcons and centrifuge with 100g for 3 min<br/>
-<div class="level3">
-<p>
-Transformation with knockdown ligations 112, 113, 114, 115, 116 and lenti-Cas9 1516 were performed according to heatshock protocol.
-</p>
 </div>
-<div class='secedit editbutton_section editbutton_295'> </div>
-<h3 class="sectionedit296" id="gel_purification_protocol">Gel purification protocol</h3>
-<div class="level3">
-<ul>
-<li class="level1"><div class="li"> add 450 µl QG buffer, 50-60°C for 10 min</div>
 </li>
-<li class="level1"><div class="li"> add 150 µl isopropanol and mix</div>
+<li class="level1"><div class="li"> Resuspend the cell pellet with 100 µl P3 Buffer<br/>
+</div>
 </li>
-<li class="level1"><div class="li"> 10 min at -20°C</div>
+<li class="level1"><div class="li"> Add 6 µg pIG17_009<br/>
+</div>
 </li>
-<li class="level1"><div class="li"> load on column</div>
+<li class="level1"><div class="li"> Transfer the mixture into cuvette <br/>
+</div>
 </li>
-<li class="level1"><div class="li"> spin for 1 min at full speed and remove flow through</div>
+<li class="level1"><div class="li"> BioRad setting: 250 V, 960 µF, 35 sec.<br/>
+</div>
 </li>
-<li class="level1"><div class="li"> wash with 750 µl PE buffer</div>
+<li class="level1"><div class="li"> Add 500 µl RPMI medium to the cells immediately after electroporation<br/>
+</div>
 </li>
-<li class="level1"><div class="li"> spin for 1 min at full speed and remove flow through</div>
+<li class="level1"><div class="li"> Plate the cells into 12-well plate with 2 ml Medium<br/>
+</div>
 </li>
-<li class="level1"><div class="li"> spin 2 min at full speed and remove flow through</div>
+<li class="level1"><div class="li"> Over night culturing<br/>
+</div>
 </li>
-<li class="level1"><div class="li"> elute DNA in 30 µl H2O</div>
+<li class="level1"><div class="li"> Wash the cuvettes for reusing<br/>
+</div>
 </li>
-</ul>
+</ol>
 </div>
-<div class='secedit editbutton_section editbutton_296'> </div>
+<div class='secedit editbutton_section editbutton_81'> </div>
-<h2 class="sectionedit297" id="section190717">19.07.17</h2>
+<h3 class="sectionedit82" id="ph_tests_with_jurkat">pH tests with Jurkat</h3>
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_297'> </div>
-<h3 class="sectionedit298" id="ligation_of_sgrna3_in_cas9-gfp_sdm_pcr_product">Ligation of sgRNA3 in Cas9-GFP SDM PCR product</h3>
 <div class="level3">
-<div class="table sectionedit299"><table class="inline">
+<div class="table sectionedit83"><table class="inline">
 	<thead>
 	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
+		<th class="col0">V(ges) [ml]</th><th class="col1">V(RPMI1640) [ml]</th><th class="col2">V(cells) [ml]</th><th class="col3">c(cells) [cells/ml]</th>
 	</tr>
 	</thead>
 	<tr class="row1">
-		<td class="col0"> 4 µl </td><td class="col1"> 8 gn/µl PCR product </td>
+		<td class="col0">7.5</td><td class="col1">5.3</td><td class="col2">2.2</td><td class="col3">250.000</td>
-	</tr>
-	<tr class="row2">
-		<td class="col0"> 4 µl </td><td class="col1"> H2O </td>
-	</tr>
-	<tr class="row3">
-		<td class="col0"> 1 µl </td><td class="col1"> T4 ligase </td>
-	</tr>
-	<tr class="row4">
-		<td class="col0"> 1 µl </td><td class="col1"> ligase buffer 10x </td>
 	</tr>
 </table></div>
-<div class='secedit editbutton_table editbutton_299'> </div>
+<div class='secedit editbutton_table editbutton_83'> </div>
 <p>
-samples 2 h at 16°C<br/>
+- incubation for 24h
-samples 1 h at 16°C<br/>
-DpnI digest (4 µl of each ligation) and heat shock frafo in XL10gold<br/>
 </p>
 </div>
-<div class='secedit editbutton_section editbutton_298'> </div>
+<div class='secedit editbutton_section editbutton_82'> </div>
-<h3 class="sectionedit300" id="minipreps_of_picked_knockdown_cultures">Minipreps of picked knockdown cultures</h3>
+<h3 class="sectionedit84" id="miniprep_of_pig_034">Miniprep of pIG_034</h3>
 <div class="level3">
 <p>
-Minipreps of knockdown plasmids 112, 113, 114, 115, 116 were performed according to manufacturer&#039;s protocol and send for sequencing.<br/>
+- miniprep 8x
 </p>
 </div>
-<div class='secedit editbutton_section editbutton_300'> </div>
+<div class='secedit editbutton_section editbutton_84'> </div>
-<h2 class="sectionedit301" id="section200717">20.07.17</h2>
+<h2 class="sectionedit85" id="section210817">21.08.17</h2>
 <div class="level2">
 </div>
-<div class='secedit editbutton_section editbutton_301'> </div>
+<div class='secedit editbutton_section editbutton_85'> </div>
-<h3 class="sectionedit302" id="picking_colonies_of_cas9-gfp_sgrna3">Picking colonies of CAS9-GFP sgRNA3</h3>
+<h3 class="sectionedit86" id="sequencing_of_pig_138">sequencing of pIG_138</h3>
 <div class="level3">
 <p>
-One colony of each plate of CAS9-GFP sgRNA3 was picked for prep, glycerol stock and digest. Plates were stored at 4°C to pick further colonies (re-plate tomorrow).
+- plasmid: CMV-VEGFR2 (pIG_138)<br/>
+- DNA concentration: 80ng/ml<br/>
+- Oligo: 2µl DNA, 18µl H2O<br/>
+- V(ges): 20µl<br/>
 </p>
 </div>
-<div class='secedit editbutton_section editbutton_302'> </div>
+<div class='secedit editbutton_section editbutton_86'> </div>
-<h2 class="sectionedit303" id="section210717">21.07.17</h2>
+<h2 class="sectionedit87" id="section220817">22.08.17</h2>
 <div class="level2">
 </div>
-<div class='secedit editbutton_section editbutton_303'> </div>
+<div class='secedit editbutton_section editbutton_87'> </div>
-<h3 class="sectionedit304" id="extraction_of_genomic_dna_from_hek293t_cells">Extraction of genomic DNA from HEK293T cells</h3>
+<h3 class="sectionedit88" id="miniprep_of_pig_017_pig_031_pig_034_pig_037">Miniprep of pIG_017, pIG_031, pIG_034, pIG_037</h3>
 <div class="level3">
 <p>
-Genomic DNA was extracted from HEK293T cells according to kit protocol for T7E1 assay establishment.
+- Zymo kit<br/>
 </p>
 </div>
-<div class='secedit editbutton_section editbutton_304'> </div>
+<div class='secedit editbutton_section editbutton_88'> </div>
-<h3 class="sectionedit305" id="positive_knockdown_colonies">Positive knockdown colonies</h3>
+<h3 class="sectionedit89" id="up-concentration_of_pig_017_pig_031_pig_034_pig_037">up-concentration of pIG_017, pIG_031, pIG_034, pIG_037</h3>
 <div class="level3">
 <p>
-Positive colonies of 114, 115, 116 (SGEN) were recultured and new colonies of 112, and 113 were picked.
+- add isopropanol to plasmid suspension in H2O<br/>
-</p>
-</div>
+- -20°C for 20min<br/>
-<div class='secedit editbutton_section editbutton_305'> </div>
-<h3 class="sectionedit306" id="miniprep_of_lenti-cas9_colonies">Miniprep of lenti-CAS9 colonies</h3>
-<div class="level3">
-<p>
+- centrifugate for 30min at maximal speed and 4°C<br/>
-A miniprep of 5 lenti-CAS colonies was performed according to manufacturer&#039;s protocol.<br/>
-</p>
+- remove isopropanol<br/>
-<p>
+- wash with EtOH (70%)<br/>
-lenti-Cas9 test digest with NcoI-HF was performed at 37°C.<br/>
-</p>
+- centrifugate for 30min att maximal speed and 4°C<br/>
-<div class="table sectionedit307"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0"> 2 µl </td><td class="col1"> DNA </td>
-	</tr>
-	<tr class="row2">
-		<td class="col0"> 0.25 µl </td><td class="col1"> NcoI-HF </td>
-	</tr>
-	<tr class="row3">
-		<td class="col0"> 1 µl </td><td class="col1"> 10x CutSmart </td>
-	</tr>
-	<tr class="row4">
-		<td class="col0"> 6.75 µl </td><td class="col1"> H2O </td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_307'> </div>
-<p>
-picture no. 534
-</p>
-<p>
+- remove EtOH<br/>
-Digest of lenti-Cas9 was performed using BsmbI.<br/>
-</p>
+- open eppis to dry overnight<br/>
-<div class="table sectionedit308"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0"> 2.5 µl </td><td class="col1"> plasmid (500 ng/µl) </td>
-	</tr>
-	<tr class="row2">
-		<td class="col0"> 5 µl </td><td class="col1"> fast digest buffer </td>
-	</tr>
-	<tr class="row3">
-		<td class="col0"> 1 µl </td><td class="col1"> BsmBI </td>
-	</tr>
-	<tr class="row4">
-		<td class="col0"> 41.5 µ </td><td class="col1"> H2O </td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_308'> </div>
-<p>
-picture no. 534
-</p>
-</div>
+- eluate with H2O<br/>
-<div class='secedit editbutton_section editbutton_306'> </div>
-<h3 class="sectionedit309" id="miniprep_of_new_picked_knockdown_colonies">Miniprep of new picked knockdown colonies</h3>
+Up-conentration did not work, DNA concentration decreased.<br/>
-<div class="level3">
-<p>
-Miniprep was performed of knockdown colonies 112 and 113 according to manufacturer&#039;s protocol.
 </p>
 </div>
-<div class='secedit editbutton_section editbutton_309'> </div>
+<div class='secedit editbutton_section editbutton_89'> </div>
-<h2 class="sectionedit310" id="section240717">24.07.17</h2>
+<h2 class="sectionedit90" id="section250817">25.08.17</h2>
 <div class="level2">
 </div>
-<div class='secedit editbutton_section editbutton_310'> </div>
+<div class='secedit editbutton_section editbutton_90'> </div>
-<h3 class="sectionedit311" id="digest_of_lenti-cas9">Digest of lenti-Cas9</h3>
+<h3 class="sectionedit91" id="miniprep_of_pig_017_pig_037">Miniprep of pIG_017, pIG_037</h3>
 <div class="level3">
-<div class="table sectionedit312"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0"> 6 µl </td><td class="col1"> plasmid (279.5 ng/µl) </td>
-	</tr>
-	<tr class="row2">
-		<td class="col0"> 5 µl </td><td class="col1"> fast digest buffer 10x </td>
-	</tr>
-	<tr class="row3">
-		<td class="col0"> 1 µl </td><td class="col1"> BsmBI </td>
-	</tr>
-	<tr class="row4">
-		<td class="col0"> 38 µl </td><td class="col1"> H2O </td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_312'> </div>
-<p>
-<img src="https://static.igem.org/mediawiki/2017/1/1b/T%E2%80%93FREIBURG%E2%80%93541.png" width="500px">
-</p>
 </div>
-<div class='secedit editbutton_section editbutton_311'> </div>
+<div class='secedit editbutton_section editbutton_91'> </div>
-<h3 class="sectionedit313" id="site_directed_mutagenesis_pcr">Site directed mutagenesis PCR</h3>
+<h2 class="sectionedit92" id="section070917">07.09.17</h2>
-<div class="level3">
-<p>
-Site directed mutagenesis PCR was perfored to obtain BsmBI and BbsI restriction sites in Cas9-GFP and BbsI in Lenti-Cas9-puro. Two fragments were generated for each insertion.<br/>
-</p>
-<div class="table sectionedit314"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">Fragment</th><th class="col1"> </th><th class="col2">1</th><th class="col3"> </th><th class="col4">2</th><th class="col5"> </th>
-	</tr>
-	<tr class="row1">
-		<th class="col0">Plasmid</th><th class="col1">Restriction site</th><th class="col2">primers</th><th class="col3">size</th><th class="col4">primers</th><th class="col5">size</th>
-	</tr>
-	</thead>
-	<tr class="row2">
-		<td class="col0"> GFP </td><td class="col1"> BsmBI </td><td class="col2"> 212/215 </td><td class="col3"> 3 kb </td><td class="col4"> 216/218 </td><td class="col5"> 3.7 kb </td>
-	</tr>
-	<tr class="row3">
-		<td class="col0"> GFP </td><td class="col1"> BbsI </td><td class="col2"> 212/214 </td><td class="col3"> 3 kb </td><td class="col4"> 216/218 </td><td class="col5"> 3.7 kb </td>
-	</tr>
-	<tr class="row4">
-		<td class="col0"> lenti </td><td class="col1"> BbsI </td><td class="col2"> 213/214 </td><td class="col3"> 1 kb </td><td class="col4"> 216/217 </td><td class="col5"> 2.9 kb </td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_314'> </div>
-<p>
-The observed fragment pattern was observed ladder was not visible on gel. Fragments were excited anyway and PCR was repeated on 25.07.17.
-</p>
-</div>
-<div class='secedit editbutton_section editbutton_313'> </div>
-<h2 class="sectionedit315" id="section250717">25.07.17</h2>
 <div class="level2">
 </div>
-<div class='secedit editbutton_section editbutton_315'> </div>
+<div class='secedit editbutton_section editbutton_92'> </div>
-<h3 class="sectionedit316" id="gel_extraction_digest_and_ligation_of_lenti-cas9">Gel extraction, digest and ligation of lenti-Cas9</h3>
+<h3 class="sectionedit93" id="pei_transfection_of_hek_cells1">PEI transfection of HEK cells</h3>
 <div class="level3">
 <p>
-lenti-CAS9 fragments were extracted from gel and digested using BsmBI.<br/>
+used cells: HEK cells<br/>
 </p>
-<div class="table sectionedit317"><table class="inline">
+<div class="table sectionedit94"><table class="inline">
 	<thead>
 	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
+		<th class="col0">Plasmid number</th><th class="col1">component</th>
 	</tr>
 	</thead>
 	<tr class="row1">
-		<td class="col0"> 6 µl </td><td class="col1"> plasmid </td>
+		<td class="col0">pIG17_034</td><td class="col1">pCRE-GFP-CMV-mCherry</td>
 	</tr>
 	<tr class="row2">
-		<td class="col0"> 5 µl </td><td class="col1"> FD 10x B. </td>
+		<td class="col0">pIG17_008 + pIG17_009</td><td class="col1">CMV-GFP + CMV-mCherry</td>
 	</tr>
 	<tr class="row3">
-		<td class="col0"> 1 µl </td><td class="col1"> BsmBI </td>
+		<td class="col0">pIG17_034 + β2-receptor</td><td class="col1">pCRE-GFP-CMV-mCherry + β2-receptor</td>
 	</tr>
 	<tr class="row4">
-		<td class="col0"> 38 µl </td><td class="col1"> H2O </td>
+		<td class="col0">no DNA</td><td class="col1">negative control</td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_317'> </div>
-<p>
-Picture no. 543<br/>
-</p>
-<p>
-Fragments of lenti-Cas9 were ligated.<br/>
-</p>
-<div class="table sectionedit318"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0"> 5 µl </td><td class="col1"> backbone (15.9 ng/µl) </td>
-	</tr>
-	<tr class="row2">
-		<td class="col0"> 1 µl </td><td class="col1"> sgRNA1, 3, 4 (50 nM)</td>
-	</tr>
-	<tr class="row3">
-		<td class="col0"> 1 µl </td><td class="col1"> T4 buffer </td>
-	</tr>
-	<tr class="row4">
-		<td class="col0"> 1 µl </td><td class="col1"> T4 </td>
-	</tr>
-	<tr class="row5">
-		<td class="col0"> 2 µl </td><td class="col1"> H2O </td>
 	</tr>
 </table></div>
-<div class='secedit editbutton_table editbutton_318'> </div>
+<div class='secedit editbutton_table editbutton_94'> </div><ol>
+<li class="level1"><div class="li"> Split the cells 1:5 into 10 cm plate<br/>
 </div>
-<div class='secedit editbutton_section editbutton_316'> </div>
+</li>
-<h3 class="sectionedit319" id="t7e1_assay">T7E1 assay</h3>
+<li class="level1"><div class="li"> Thaw PEI reagent at RT<br/>
-<div class="level3">
-<p>
-NEB 2 puffer 1x and 1.5 µl T7 were added to 200 ng of PCR products (of positive control, exon 6, exon 5, exon 8, exon 10). Assay was cooled down in 2.5 h from 95°C to 20°C.
-</p>
 </div>
-<div class='secedit editbutton_section editbutton_319'> </div>
+</li>
-<h3 class="sectionedit320" id="miniprep_of_sgen_115_and_lenti-cas9_plasmids">Miniprep of SGEN 115 and lenti-Cas9 plasmids</h3>
+<li class="level1"><div class="li"> Dilute plasmid in 1 ml serum free DMEM (2.5 µg from each plasmid)<br/>
-<div class="level3">
-<p>
-Minipreps were performed according to manufacturer&#039;s protocol.
-</p>
 </div>
-<div class='secedit editbutton_section editbutton_320'> </div>
+</li>
-<h2 class="sectionedit321" id="section260717">26.07.17</h2>
+<li class="level1"><div class="li"> Add 15 µl PEI(1 µg/µl) to the mix while vortexing (PEI:DNA = 3:1)<br/>
-<div class="level2">
 </div>
-<div class='secedit editbutton_section editbutton_321'> </div>
+</li>
-<h3 class="sectionedit322" id="picking_colonies_for_lenti-cas9">Picking colonies for lenti-Cas9</h3>
+<li class="level1"><div class="li"> incubate 15 min at RT<br/>
-<div class="level3">
-<p>
-Colonies containing sgRNA1, 3, 4, 5, 9 in lenti-CAS9 were picked.<br/>
-</p>
 </div>
-<div class='secedit editbutton_section editbutton_322'> </div>
+</li>
-<h3 class="sectionedit323" id="digest_of_lenti-cas9_with_bsmbi6">Digest of lenti-Cas9 with BsmBI</h3>
+<li class="level1"><div class="li"> Add the mix to the culture<br/>
-<div class="level3">
-<div class="table sectionedit324"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0"> 3 µl </td><td class="col1"> plasmid </td>
-	</tr>
-	<tr class="row2">
-		<td class="col0"> 2.5 µl </td><td class="col1"> FD buffer </td>
-	</tr>
-	<tr class="row3">
-		<td class="col0"> 0.5 µl </td><td class="col1"> BsmBI </td>
-	</tr>
-	<tr class="row4">
-		<td class="col0"> 19 µl </td><td class="col1"> H2O </td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_324'> </div>
-<p>
-picture no. 547
-</p>
 </div>
-<div class='secedit editbutton_section editbutton_323'> </div>
+</li>
-<h3 class="sectionedit325" id="sdm_pcr_bsmbi_and_bbsi_into_cas9-gfp_and_lenti-cas9-puro">SDM PCR BsmBI and BbsI into Cas9-GFP and lenti-Cas9-puro</h3>
+<li class="level1"><div class="li"> incubate for 24 h<br/>
-<div class="level3">
-<p>
-Site-directed mutagenesis was performed as previously to insert BsmBI and BbsI into Cas9-GFP. PCR fragments were excised from gel and purified using a clear-up kit according to manufacturer&#039;s protocol. Fragments were digested with BsmBI or BbsI and NotI or EcoRI and purified using a PCR clear-up kit.<br/>
-Cas9-GFP and lenti-Cas9-puro were digested with NotI and EcoRI in FD 10x.<br/>
-picutres no. 539 and 538<br/>
-</p>
 </div>
-<div class='secedit editbutton_section editbutton_325'> </div>
+</li>
-<h2 class="sectionedit326" id="section270717">27.07.17</h2>
+</ol>
-<div class="level2">
 </div>
-<div class='secedit editbutton_section editbutton_326'> </div>
+<div class='secedit editbutton_section editbutton_93'> </div>
-<h3 class="sectionedit327" id="site_directed_mutagenesis_pcr1">Site directed mutagenesis PCR</h3>
+<h3 class="sectionedit95" id="gel_extraction_of_m1-receptor_without_cfp">Gel extraction of M1-receptor without CFP</h3>
 <div class="level3">
 <p>
-Strategy was changed because strategy with shorter fragments for each side combined with NotI and EcoRI did not work (produced blunt-ends). Now a whole plasmid amplification was performed to insert BsmBI and BbsI into Cas9-GFP (run on 0.9% agarose at 110 V for 35 min, picture no. IMG00550). Protocols as for oroginal blunt end ligation estrategy, expected band of 9.3 kb was at 10 kb with good efficiency, also a 3 kb band was observed and both were extracted.<br/>
+ng DNA<br/>
-</p>
+µl T4 Ligase<br/>
-</div>
+µl Buffer 10x<br/>
-<div class='secedit editbutton_section editbutton_327'> </div>
-<h3 class="sectionedit328" id="digest_with_bsmbi_or_bbsi">Digest with BsmBI or BbsI</h3>
-<div class="level3">
-<div class="table sectionedit329"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0"> 0.5 µl </td><td class="col1"> BsmBI </td>
-	</tr>
-	<tr class="row2">
-		<td class="col0 leftalign">  </td><td class="col1"> fermentas FD 10x </td>
-	</tr>
-	<tr class="row3">
-		<td class="col0"> 1 µl </td><td class="col1"> DpnI </td>
-	</tr>
-	<tr class="row4">
-		<td class="col0 leftalign">  </td><td class="col1"> H2O </td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_329'> </div>
-<p>
-Digest was performed for 20 min at 37 °C and 1 h at 55°C.<br/>
-</p>
+µl H2O<br/>
-<div class="table sectionedit330"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0"> 1 µl </td><td class="col1"> BbsI </td>
-	</tr>
-	<tr class="row2">
-		<td class="col0 leftalign">  </td><td class="col1"> fermentas FD 10x </td>
-	</tr>
-	<tr class="row3">
-		<td class="col0"> 1 µl </td><td class="col1"> DpnI </td>
-	</tr>
-	<tr class="row4">
-		<td class="col0 leftalign">  </td><td class="col1"> H2O </td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_330'> </div>
-<p>
-Digest was performed for 2 h at 37°C.<br/>
-</p>
-<p>
-Oligo ligation of sgRNA5 with T4 was performed for 1 h and XL10gold were transformed according to heat shock protocol. Colonies were also found in BbsI (maybe confused) and BsmpI (DpnI might need longer).<br/>
 </p>
 </div>
-<div class='secedit editbutton_section editbutton_328'> </div>
+<div class='secedit editbutton_section editbutton_95'> </div>
-<h3 class="sectionedit331" id="miniprep_of_lenti-cas92">Miniprep of Lenti-Cas9</h3>
+<h2 class="sectionedit96" id="section080917">08.09.17</h2>
-<div class="level3">
-<p>
-Minipreps of plasmids containing sgRNA1, 3, 4, 5, 9 were performed according to manufacturer&#039;s protocol and send for sequencing.
-</p>
-</div>
-<div class='secedit editbutton_section editbutton_331'> </div>
-<h2 class="sectionedit332" id="section280717">28.07.17</h2>
 <div class="level2">
 </div>
-<div class='secedit editbutton_section editbutton_332'> </div>
+<div class='secedit editbutton_section editbutton_96'> </div>
-<h3 class="sectionedit333" id="gelex_of_digested_lenti-cas9_and_ligation_of_lenti-cas_sgrna20">Gelex of digested lenti-CAS9 and ligation of lenti-Cas sgRNA20</h3>
+<h3 class="sectionedit97" id="colony_pcr_of_m1">Colony PCR of M1</h3>
 <div class="level3">
-<div class="table sectionedit334"><table class="inline">
+<div class="table sectionedit98"><table class="inline">
-	<thead>
 	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
+		<td class="col0">Buffer 10x</td><td class="col1">2.5µl</td><td class="col2">12.5 µl</td>
 	</tr>
-	</thead>
 	<tr class="row1">
-		<td class="col0"> 7 µl </td><td class="col1"> plasmid </td>
+		<td class="col0">dNTP 10 mM</td><td class="col1">0.5 µl</td><td class="col2">2.5 µl</td>
 	</tr>
 	<tr class="row2">
-		<td class="col0"> 1 µl </td><td class="col1"> sgRNA (50 ng/µl)</td>
+		<td class="col0">Template</td><td class="col1">1 µl</td><td class="col2"> </td>
 	</tr>
 	<tr class="row3">
-		<td class="col0"> 1 µl </td><td class="col1"> T4 ligase buffer </td>
+		<td class="col0">Primer</td><td class="col1">0.5 µl</td><td class="col2">2.5 µl</td>
 	</tr>
 	<tr class="row4">
-		<td class="col0"> 1 µl </td><td class="col1"> T4 ligase </td>
+		<td class="col0">DreamTaq Thermo Fisher</td><td class="col1">0.25 µl</td><td class="col2">1.25 µl</td>
+	</tr>
+	<tr class="row5">
+		<td class="col0">H2O</td><td class="col1">20 µl</td><td class="col2">100 µl</td>
 	</tr>
 </table></div>
-<div class='secedit editbutton_table editbutton_334'> </div>
+<div class='secedit editbutton_table editbutton_98'> </div>
 </div>
-<div class='secedit editbutton_section editbutton_333'> </div>
+<div class='secedit editbutton_section editbutton_97'> </div>
-<h3 class="sectionedit335" id="pcr_for_t7e1">PCR for T7E1</h3>
+<h2 class="sectionedit99" id="section2008171">20.08.17</h2>
-<div class="level3">
-<p>
-PCR for T7E1 was performed to identify knockout in HEK cells as previously described. PCR purification was performed of HEK both primers and exon 8.
-</p>
-</div>
-<div class='secedit editbutton_section editbutton_335'> </div>
-<h2 class="sectionedit336" id="section300717">30.07.17</h2>
 <div class="level2">
 </div>
-<div class='secedit editbutton_section editbutton_336'> </div>
+<div class='secedit editbutton_section editbutton_99'> </div>
-<h3 class="sectionedit337" id="pcr_for_t7e1_assay">PCR for T7E1 assay</h3>
+<h3 class="sectionedit100" id="pcre_induction">pCRE induction</h3>
 <div class="level3">
 <p>
-Positive control: CCR5delta32 mutation in HEK293T (olG17_073/074).<br/>
+- treatment in 6-well-plate<br/>
-Negative sample: HIF1A exon 8.<br/>
+- add Forskolin and β2-inductor to the cells<br/>
 </p>
-<div class="table sectionedit338"><table class="inline">
+<p>
+<strong>Cells transfected with pIG17_034 (pCRE-GFP-CMV-mCherry):</strong>
+</p>
+<div class="table sectionedit101"><table class="inline">
 	<thead>
 	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
+		<th class="col0">approach</th><th class="col1">Forskolin</th><th class="col2">β2-inductor</th>
 	</tr>
 	</thead>
 	<tr class="row1">
-		<td class="col0"> x µl </td><td class="col1"> gDNA (300ng)</td>
+		<td class="col0">negative control</td><td class="col1">-</td><td class="col2">-</td>
 	</tr>
 	<tr class="row2">
-		<td class="col0"> 5 µl </td><td class="col1"> 5x phusion HF buffer </td>
+		<td class="col0">old Forskolin</td><td class="col1">10 µM (old)</td><td class="col2">-</td>
 	</tr>
 	<tr class="row3">
-		<td class="col0"> 0.5 µl </td><td class="col1"> dNTPs</td>
+		<td class="col0">new Forskolin</td><td class="col1">10 µM (new)</td><td class="col2">-</td>
 	</tr>
 	<tr class="row4">
-		<td class="col0"> 1 µl </td><td class="col1"> fwd </td>
+		<td class="col0">new Forskolin</td><td class="col1">100 µM new Forskolin</td><td class="col2"></td>
 	</tr>
 	<tr class="row5">
-		<td class="col0"> 1 µl </td><td class="col1"> rev </td>
+		<td class="col0">β2-inductor</td><td class="col1">-</td><td class="col2">10 µM</td>
 	</tr>
 	<tr class="row6">
-		<td class="col0"> 0.25 µl </td><td class="col1"> phusion</td>
+		<td class="col0">β2-inductor</td><td class="col1">-</td><td class="col2">100 µM</td>
-	</tr>
-	<tr class="row7">
-		<td class="col0"> 17.25-x µl </td><td class="col1"> H2O </td>
 	</tr>
 </table></div>
-<div class='secedit editbutton_table editbutton_338'> </div>
+<div class='secedit editbutton_table editbutton_101'> </div>
 <p>
-(original protocol: 200 ng gDNA)<br/>
+<strong>Cells transfected with pIG17_034 (pCRE-GFP-CMV-mCherry) and β2-receptor:
+</strong>
-Better use 0.5 µl primers for 25 µl reactions.<br/>
-Electrophoresis was performed on 2% agarose gel (100 V for 45 min).<br/>
-Expected bands: 500 bp<br/>
 </p>
+<div class="table sectionedit102"><table class="inline">
-</div>
-<div class='secedit editbutton_section editbutton_337'> </div>
-<h3 class="sectionedit339" id="pcr_hek_positive_control_73_74_test">PCR HEK positive control 73,74 test</h3>
-<div class="level3">
-<div class="table sectionedit340"><table class="inline">
 	<thead>
 	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
+		<th class="col0">approach</th><th class="col1">Forskolin</th><th class="col2">β2-inductor</th>
 	</tr>
 	</thead>
 	<tr class="row1">
-		<td class="col0"> 8 µl </td><td class="col1"> H2O </td>
+		<td class="col0">negative control</td><td class="col1">-</td><td class="col2">-</td>
 	</tr>
 	<tr class="row2">
-		<td class="col0"> 12,5 µl </td><td class="col1"> kappa2G </td>
+		<td class="col0">β2-inductor</td><td class="col1">-</td><td class="col2">10 µM</td>
 	</tr>
 	<tr class="row3">
-		<td class="col0"> 1.25 µl </td><td class="col1"> primer 73 </td>
+		<td class="col0">β2-inductor</td><td class="col1">-</td><td class="col2">100 µM</td>
-	</tr>
-	<tr class="row4">
-		<td class="col0"> 1.25 µl </td><td class="col1"> primer 74 </td>
 	</tr>
 </table></div>
-<div class='secedit editbutton_table editbutton_340'> </div>
+<div class='secedit editbutton_table editbutton_102'> </div>
 </div>
-<div class='secedit editbutton_section editbutton_339'> </div>
+<div class='secedit editbutton_section editbutton_100'> </div>
-<h2 class="sectionedit341" id="section310717">31.07.17</h2>
-<div class="level2">
 </div>
-<div class='secedit editbutton_section editbutton_341'> </div>
+<button class="accordion">September</button>
-<h3 class="sectionedit342" id="sequencing_of_cas9-gfp_sgrnas">Sequencing of Cas9-GFP sgRNAs</h3>
+         <div class="panel panel-default">
-<div class="level3">
+<h2 class="sectionedit103" id="section100917">10.09.17</h2>
-<p>
+<div class="level2">
-Cas9-GFP sgRNAs1, 3, 4, 9, 20 from SDM PCR restriction site insertion, BsmBI or BbsI and DpnI digested and dsDNA oligoinsertion were send to sequencing.
-</p>
 </div>
-<div class='secedit editbutton_section editbutton_342'> </div>
+<div class='secedit editbutton_section editbutton_103'> </div>
-<h3 class="sectionedit343" id="pcr_purification_of_t7e1_samples">PCR purification of T7E1 samples</h3>
+<h3 class="sectionedit104" id="pei_transfection_of_plasmids_pig17_022_23_37_861">PEI transfection of plasmids pIG17_022/23/37/86</h3>
 <div class="level3">
 <p>
-T7E1 dsDNA samples obtained on 30.07.17 were dissociated and re-annealed in NEB 2 buffer 1x at 95°C for 5 min and for 2:30 h slowly  cooled down to room temperature in heatblock.
+used cells: HEK cells<br/>
-</p>
-</div>
-<div class='secedit editbutton_section editbutton_343'> </div>
-<h3 class="sectionedit344" id="t7e1_assay1">T7E1 assay</h3>
-<div class="level3">
-<p>
-ng PCR product were re-annealed using 1.5 µl 1:1 of T7E1 and and 2x NEB 2 buffer at 37°C for less than 20 min. Reaction was stopped by addition of 3 µl 6x OrangeDye, the samples were put on ice immediately and quickly loaded on 2% agarose gel.<br/>
-Expected fragments: 200 bp, 300 bp, could not be observed.
 </p>
+<div class="table sectionedit105"><table class="inline">
+	<thead>
+	<tr class="row0">
+		<th class="col0">Plasmid number</th><th class="col1">component</th>
+	</tr>
+	</thead>
+	<tr class="row1">
+		<td class="col0">pIG17_031</td><td class="col1">HRE-GFP-CMV-mCherry</td>
+	</tr>
+</table></div>
+<div class='secedit editbutton_table editbutton_105'> </div><ol>
+<li class="level1"><div class="li"> Split the cells 1:2 into 10 cm plate<br/>
 </div>
-<div class='secedit editbutton_section editbutton_344'> </div>
+</li>
-              </div>
+<li class="level1"><div class="li"> Thaw PEI reagent at RT<br/>
-              <button class="accordion">August</button>
-        <div id="vanish" class="panel panel-default">
-<h2 class="sectionedit345" id="section010817">01.08.17</h2>
-<div class="level2">
 </div>
-<div class='secedit editbutton_section editbutton_345'> </div>
+</li>
-<h3 class="sectionedit346" id="knockout_sequencing">Knockout Sequencing</h3>
+<li class="level1"><div class="li"> Dilute plasmid in 1 ml serum free DMEM (2.5 µg from each plasmid)<br/>
-<div class="level3">
-<p>
-Cas9-GFP sgRNAs from 31.07.17, only positive for BsmBI samples<br/>
-repeat of SDM PCR with Q5 to insert BsmBI sites into Cas9-GFP<br/>
-sequencing of other colonies for sgRNAs 4, 9 inserted by BsmBI, overnight cultures for miniprep
-</p>
 </div>
-<div class='secedit editbutton_section editbutton_346'> </div>
-<h3 class="sectionedit347" id="t7e1_assay_repeat_troubleshooting">T7E1 assay repeat troubleshooting</h3>
-<div class="level3">
-<ul>
-<li class="level1"><div class="li"> use more DNA</div>
 </li>
-<li class="level1"><div class="li"> anneal longer than 3 h</div>
+<li class="level1"><div class="li"> Add 15 µl PEI(1 µg/µl) to the mix while vortexing (PEI:DNA = 3:1)<br/>
+</div>
 </li>
-<li class="level1"><div class="li"> T7 less than 15 min</div>
+<li class="level1"><div class="li"> incubate 15 min at RT<br/>
+</div>
 </li>
-</ul>
+<li class="level1"><div class="li"> Add the mix to the culture<br/>
 </div>
-<div class='secedit editbutton_section editbutton_347'> </div>
+</li>
-<h3 class="sectionedit348" id="t7e1_pcr">T7E1 PCR</h3>
+<li class="level1"><div class="li"> incubate for 24 h<br/>
-<div class="level3">
-<p>
-x at 63°C, no good result on first gel
-</p>
 </div>
-<div class='secedit editbutton_section editbutton_348'> </div>
+</li>
-<h2 class="sectionedit349" id="section020817">02.08.17</h2>
+</ol>
-<div class="level2">
 </div>
-<div class='secedit editbutton_section editbutton_349'> </div>
+<div class='secedit editbutton_section editbutton_104'> </div>
-<h3 class="sectionedit350" id="miniprep_of_cas9-gfp_sgrna4_9">Miniprep of Cas9-GFP sgRNA4/9</h3>
+<h3 class="sectionedit106" id="hre_induction">HRE induction</h3>
 <div class="level3">
 <p>
-more colonies for each BsmBI cloning (27.07.17) because sequencing was negative<br/>
+- add CoCl2 (end concentrations: 0 µM, 5 µM, 10 µM, 20 µM, 50 µM, 100 µM, 200 µM, 400 µM, 800 µM)<br/>
-picking 3 colonies from BsmBI cloning (01.08.17) for sgRNAs3/20 in Cas9-GFP, other plates stored<br/>
+- fluoresence microscopy<br/>
-control transformation yielded no colonies this time, maybe because of longer digestion with DpnI<br/>
-ml overnight cultures (2x each)
 </p>
-<ul>
-<li class="level1"><div class="li"> lenti-Cas9-puro sgRNas 1 / 3 / 4 at 37°C</div>
-</li>
-<li class="level1"><div class="li"> SGEN shRNA 4 (former 115, now 133) and shRNA 5 (former 116, now 134) at 30°C</div>
-</li>
-</ul>
 </div>
-<div class='secedit editbutton_section editbutton_350'> </div>
+<div class='secedit editbutton_section editbutton_106'> </div>
-<h3 class="sectionedit351" id="t7e1_pcr1">T7E1 PCR</h3>
+<h3 class="sectionedit107" id="repetition_of_the_interlab_study">Repetition of the Interlab Study</h3>
 <div class="level3">
 <p>
-PCR at 60°C worked with HEK but not wich Ex8.<br/>
+- transformation of DH5-α with plasmids from Kit 7 (21B, 21D, 21F, 21H, 21L, 21N, 21P)<br/>
-PCR purification HEK (30 ng) and Ex8 (15 ng).<br/>
+- incubation at 37 °C<br/>
 </p>
-<p>
+</div>
-Standard PCR protocol HEK 73/74
+<div class='secedit editbutton_section editbutton_107'> </div>
-</p>
+<h3 class="sectionedit108" id="pei_transfection_of_hek_cells2">PEI transfection of HEK cells</h3>
-<div class="table sectionedit352"><table class="inline">
+<div class="level3">
+<div class="table sectionedit109"><table class="inline">
 	<thead>
 	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
+		<th class="col0">Plasmid number</th><th class="col1">component</th>
 	</tr>
 	</thead>
 	<tr class="row1">
-		<td class="col0"> 0.5 µl </td><td class="col1"> DNA (200 ng)</td>
+		<td class="col0">pIG17_034 + L3 (Luciferase)</td><td class="col1">pCRE-GFP-CMV-mCherry + L3</td>
 	</tr>
 	<tr class="row2">
-		<td class="col0"> 5 µl </td><td class="col1"> phusion 5x buffer </td>
+		<td class="col0">pIG17_034 + TDAG8</td><td class="col1">pCRE-GFP-CMV-mCherry + TDAG8</td>
 	</tr>
 	<tr class="row3">
-		<td class="col0"> 0.5 µl </td><td class="col1"> dNTPs </td>
+		<td class="col0">pIG17_008 + pIG17_009</td><td class="col1">CMV-GFP + CMV-mCherry</td>
-	</tr>
-	<tr class="row4">
-		<td class="col0"> 0.5 µl </td><td class="col1"> fwd </td>
-	</tr>
-	<tr class="row5">
-		<td class="col0"> 0.5 µl </td><td class="col1"> rev </td>
-	</tr>
-	<tr class="row6">
-		<td class="col0"> 0.25 µl </td><td class="col1"> phusion </td>
-	</tr>
-	<tr class="row7">
-		<td class="col0"> 17.75 µl </td><td class="col1"> H2O </td>
 	</tr>
 </table></div>
-<div class='secedit editbutton_table editbutton_352'> </div>
+<div class='secedit editbutton_table editbutton_109'> </div>
 </div>
-<div class='secedit editbutton_section editbutton_351'> </div>
+<div class='secedit editbutton_section editbutton_108'> </div>
-<h2 class="sectionedit353" id="section030817">03.08.17</h2>
+<h2 class="sectionedit110" id="section160917">16.09.17</h2>
 <div class="level2">
 </div>
-<div class='secedit editbutton_section editbutton_353'> </div>
+<div class='secedit editbutton_section editbutton_110'> </div>
-<h3 class="sectionedit354" id="t7e1_annealing">T7E1 annealing</h3>
+<h3 class="sectionedit111" id="pick_colonies_of_interlab_study">Pick colonies of Interlab Study</h3>
 <div class="level3">
 <p>
-HEK CCR6 PCR product, 1 h,2 h, and 6 h at 37°C, no negative control<br/>
+- 2 colonies per plate<br/>
+- incubation at 37°C<br/>
-T7E1 incubation for 29°C at 37°C
 </p>
 </div>
-<div class='secedit editbutton_section editbutton_354'> </div>
+<div class='secedit editbutton_section editbutton_111'> </div>
-<h3 class="sectionedit355" id="minipreps_and_glycerol_stocks">Minipreps and Glycerol stocks</h3>
+<h3 class="sectionedit112" id="pcre_induction_in_hek_cells">pCRE induction in HEK cells</h3>
 <div class="level3">
 <p>
-low yield minipreps<br/>
+- plate out cells on 24-well-dish<br/>
-sgRNA1, 3, 4 in Lenti-Cas9<br/>
+- treatment with Forskolin<br/>
-sgRNA3, 20 in Cas9-GFP<br/>
-sgRNA1 in Cas9-GFP<br/>
-new overnight cultures 9 ml Cas9-GFP sgRNA1, 4, 5, 2 colonies of Cas9<br/>
-miniprep (promega) of lenti-Cas9 colonies
 </p>
+<div class="table sectionedit113"><table class="inline">
-</div>
-<div class='secedit editbutton_section editbutton_355'> </div>
-<h2 class="sectionedit356" id="section040817">04.08.17</h2>
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_356'> </div>
-<h3 class="sectionedit357" id="pcr_on_gdna_for_t7">PCR on gDNA for T7</h3>
-<div class="level3">
-<p>
-New primers for exon 9, 10 in Hif1A improved PCR, old primers for exon 6, 8 worked fine. Annealing at 60°C annealing lead to weak band for exon 6. No bands were observed for CCR5delta 32 in HEK (bad gDNA was used, now thrown away). Experiment for CCRDdelta32 was repeated with good gDNA(59°C annealing) to obtain new samples.
-</p>
-</div>
-<div class='secedit editbutton_section editbutton_357'> </div>
-<h3 class="sectionedit358" id="pcr_on_gdna_of_jurkat_cells_with_ko-kit_primers">PCR on gDNA of Jurkat cells with KO-Kit primers</h3>
-<div class="level3">
-<div class="table sectionedit359"><table class="inline">
 	<thead>
 	<tr class="row0">
-		<th class="col0">volume</th><th class="col1"> </th><th class="col2">component</th>
+		<th class="col0">Plasmid number</th><th class="col1">component</th><th class="col2">Forskolin treatment</th>
 	</tr>
 	</thead>
 	<tr class="row1">
-		<td class="col0"> 0.2 µl </td><td class="col1"> 1.8 µl </td><td class="col2"> Taq </td>
+		<td class="col0">pIG17_008 + pIG17_009</td><td class="col1">CMV-GFP + CMV-mCherry</td><td class="col2">-</td>
 	</tr>
 	<tr class="row2">
-		<td class="col0"> 1.5 µl </td><td class="col1"> 13.5 µl </td><td class="col2"> buffer </td>
+		<td class="col0">pIG17_034 + TDAG8</td><td class="col1">pCRE-GFP-CMV-mCherry + TDAG8</td><td class="col2">+</td>
 	</tr>
 	<tr class="row3">
-		<td class="col0"> 1 µl </td><td class="col1"> 9 µl </td><td class="col2"> template </td>
+		<td class="col0">no DNA</td><td class="col1">-</td><td class="col2">-</td>
 	</tr>
 	<tr class="row4">
-		<td class="col0"> 0.3 µl </td><td class="col1"> 2.7 µl </td><td class="col2"> dNTPs </td>
+		<td class="col0">no DNA</td><td class="col1">-</td><td class="col2">+</td>
 	</tr>
 	<tr class="row5">
-		<td class="col0"> 0.75 µl </td><td class="col1"> 6.75 µl </td><td class="col2"> fw </td>
+		<td class="col0">pIG17_034</td><td class="col1">pCRE-GFP-CMV-mCherry</td><td class="col2">-</td>
 	</tr>
 	<tr class="row6">
-		<td class="col0"> 0.75 µl </td><td class="col1"> 6.75 µl </td><td class="col2"> rv </td>
+		<td class="col0">pIG17_034</td><td class="col1">pCRE-GFP-CMV-mCherry</td><td class="col2">+</td>
-	</tr>
-	<tr class="row7">
-		<td class="col0"> 10.5 µl </td><td class="col1"> 95.5 µl </td><td class="col2"> H2O </td>
 	</tr>
 </table></div>
-<div class='secedit editbutton_table editbutton_359'> </div>
+<div class='secedit editbutton_table editbutton_113'> </div>
 <p>
-PCR gDNA in was annealed in thermocycler.
+- after 10 hours: pH change to 6.5, 7.0, 7.5<br/>
-</p>
-<ul>
-<li class="level1"><div class="li"> 95-85°C at 2°C/min</div>
-</li>
-<li class="level1"><div class="li"> 85-25°C at 0.05°C/min</div>
-</li>
-</ul>
-</div>
+- plate reader after 1h, 3h, 6h, 12h, 24h (Ex.: 488±20 nm; Emm.: 509±20 nm)<br/>
-<div class='secedit editbutton_section editbutton_358'> </div>
-<h3 class="sectionedit360" id="t7e1_endonuclease_assay">T7E1 endonuclease assay</h3>
-<div class="level3">
-<p>
-In Jurkat cells 200 ng gDNA were annealed (HIF1A 1.5 µl 1:1 T7:NEB2 2x, exon 6). No control was used.<br/>
-In HEK293T cells 200 ng gDNA were annealed (CCR5delta32 1.5, 2, 2.5, 3, 4 used 1:1 T7:NEB2). Control was used.<br/>
+- FACS analysis after 24h<br/>
-Annealing was performed at 37°C for 25 min.
 </p>
-</div>
-<div class='secedit editbutton_section editbutton_360'> </div>
-<h3 class="sectionedit361" id="minipreps_cas9-gfp">Minipreps Cas9-GFP</h3>
-<div class="level3">
 <p>
-Minipreps of Cas9-GFP sgRNA1, 4, 5 were performed for electroporation according to manufacturer&#039;s protocol.
+<strong><em class="u">FACS</em></strong>
 </p>
+<div class="table sectionedit112"><table class="inline">
-</div>
-<div class='secedit editbutton_section editbutton_361'> </div>
-<h2 class="sectionedit362" id="section050817">05.08.17</h2>
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_362'> </div>
-<h3 class="sectionedit363" id="t7e1_annealing1">T7E1 annealing</h3>
-<div class="level3">
-<p>
-HEK
-</p>
-<div class="table sectionedit364"><table class="inline">
-	<thead>
 	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
+		<td class="col0"><img style="max-height: 300px" src="https://static.igem.org/mediawiki/2017/2/2d/T-Freiburg-9_16_2017_pCRE_induction_1.png"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=6-5_7-0_7-5.png" class="media" title="6-5_7-0_7-5.png"><img src="/igem2017/lib/exe/fetch.php?w=200&amp;tok=ce1efc&amp;media=6-5_7-0_7-5.png" class="mediacenter" alt="" width="200" /></a></td><td class="col1"><img style="max-height: 300px" src="https://static.igem.org/mediawiki/2017/2/26/T-Freiburg-9_16_2017_pCRE_induction_2.png"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=ph_6-5.png" class="media" title="ph_6-5.png"><img src="/igem2017/lib/exe/fetch.php?w=200&amp;tok=5db4c2&amp;media=ph_6-5.png" class="mediacenter" alt="" width="200" /></a></td>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0 rightalign">  4 µl </td><td class="col1"> DNA (200ng) </td>
-	</tr>
-	<tr class="row2">
-		<td class="col0"> 0.75 µl </td><td class="col1"> T7 </td>
-	</tr>
-	<tr class="row3">
-		<td class="col0"> 0.75 µl </td><td class="col1"> NEB2 2x </td>
-	</tr>
-	<tr class="row4">
-		<td class="col0"> 8 µl </td><td class="col1"> NEB2 1x </td>
 	</tr>
 </table></div>
-<div class='secedit editbutton_table editbutton_364'> </div>
+<div class='secedit editbutton_table editbutton_112'> </div>
 <p>
-Positive control
+<strong><em class="u">Plate Reader</em></strong>
 </p>
-<div class="table sectionedit365"><table class="inline">
+<div class="table sectionedit113"><table class="inline">
-	<thead>
 	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
+		<td class="col0"><img style="max-height: 300px" src="https://static.igem.org/mediawiki/2017/0/0e/T-Freiburg-9_16_2017_pCRE_induction_3.png"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=6-5_7-0_7-5_pr.png" class="media" title="6-5_7-0_7-5_pr.png"><img src="/igem2017/lib/exe/fetch.php?w=200&amp;tok=509f4a&amp;media=6-5_7-0_7-5_pr.png" class="mediacenter" alt="" width="200" /></a></td><td class="col1"><img style="max-height: 300px" src="https://static.igem.org/mediawiki/2017/e/e7/T-Freiburg-9_16_2017_pCRE_induction_4.png"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=6-5_pr.png" class="media" title="6-5_pr.png"><img src="/igem2017/lib/exe/fetch.php?w=200&amp;tok=6a416f&amp;media=6-5_pr.png" class="mediacenter" alt="" width="200" /></a></td>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0 rightalign">  4 µl </td><td class="col1"> gDNA (58 ng/µl) </td>
-	</tr>
-	<tr class="row2">
-		<td class="col0"> 0.75 µl </td><td class="col1"> T7 </td>
-	</tr>
-	<tr class="row3">
-		<td class="col0"> 0.75 µl </td><td class="col1"> NEB2 2x </td>
-	</tr>
-	<tr class="row4">
-		<td class="col0"> 8 µl </td><td class="col1"> NEB2 1x </td>
 	</tr>
 </table></div>
-<div class='secedit editbutton_table editbutton_365'> </div>
+<div class='secedit editbutton_table editbutton_113'> </div>
-<p>
+</div>
-Negative control
+<div class='secedit editbutton_section editbutton_110'> </div>
-</p>
+<h2 class="sectionedit114" id="section170917">17.09.17</h2>
-<div class="table sectionedit366"><table class="inline">
+<div class="level2">
-	<thead>
-	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0 rightalign">  4 µl </td><td class="col1"> gDNA exon 5 (25 ng/µl) </td>
-	</tr>
-	<tr class="row2">
-		<td class="col0"> 0.75 µl </td><td class="col1"> T7 </td>
-	</tr>
-	<tr class="row3">
-		<td class="col0"> 0.75 µl </td><td class="col1"> NEB2 2x </td>
-	</tr>
-	<tr class="row4">
-		<td class="col0"> 8 µl </td><td class="col1"> NEB2 1x </td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_366'> </div>
-<p>
-Gel at 95V for 50 min.
-</p>
 </div>
-<div class='secedit editbutton_section editbutton_363'> </div>
+<div class='secedit editbutton_section editbutton_114'> </div>
-<h3 class="sectionedit367" id="pcr_ko-kit_confirm">PCR KO-Kit confirm</h3>
+<h3 class="sectionedit115" id="pei_transfection_of_plasmid_pig17_031">PEI transfection of plasmid pIG17_031</h3>
 <div class="level3">
 <p>
-PCR was performed to confirm knockout.
+used cells: HEK cells<br/>
 </p>
-<div class="table sectionedit368"><table class="inline">
+<div class="table sectionedit116"><table class="inline">
 	<thead>
 	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
+		<th class="col0">Plasmid number</th><th class="col1">component</th>
 	</tr>
 	</thead>
 	<tr class="row1">
-		<td class="col0"> 0.2 µl </td><td class="col1"> taq </td>
+		<td class="col0">pIG17_031</td><td class="col1">HRE-GFP-CMV-mCherry</td>
 	</tr>
 	<tr class="row2">
-		<td class="col0"> 2 µl </td><td class="col1"> template </td>
+		<td class="col0">pIG17_008 + pIG17_009</td><td class="col1">CMV-GFP + CMV-mCherry</td>
-	</tr>
-	<tr class="row3">
-		<td class="col0"> 1.5 µl </td><td class="col1"> 10x buffer </td>
-	</tr>
-	<tr class="row4">
-		<td class="col0"> 0.3 µl </td><td class="col1"> dNTPs </td>
-	</tr>
-	<tr class="row5">
-		<td class="col0"> 0.75 µl </td><td class="col1"> fw (32) </td>
-	</tr>
-	<tr class="row6">
-		<td class="col0"> 0.75 µl </td><td class="col1"> rv (59) </td>
-	</tr>
-	<tr class="row7">
-		<td class="col0"> 9.5 µl </td><td class="col1"> H2O </td>
 	</tr>
 </table></div>
-<div class='secedit editbutton_table editbutton_368'> </div>
+<div class='secedit editbutton_table editbutton_116'> </div><ol>
+<li class="level1"><div class="li"> Split the cells 1:2 into 10 cm plate (on 16.09.17)<br/>
 </div>
-<div class='secedit editbutton_section editbutton_367'> </div>
-<h3 class="sectionedit369" id="glycerol_stocks_cas9-gfp">Glycerol stocks Cas9-GFP</h3>
-<div class="level3">
-<p>
-Cas9-GFP BsmbI
-</p>
-<ul>
-<li class="level1"><div class="li"> sgRNA20 colonies 4, 5 and 6</div>
 </li>
-<li class="level1"><div class="li"> sgRNA3 colonies 4, 5 and 6</div>
+<li class="level1"><div class="li"> Thaw PEI reagent at RT<br/>
+</div>
 </li>
-</ul>
+<li class="level1"><div class="li"> Dilute plasmid in 1 ml serum free DMEM (2.5 µg from each plasmid)<br/>
 </div>
-<div class='secedit editbutton_section editbutton_369'> </div>
+</li>
-<h3 class="sectionedit370" id="test_digest_of_sgen112_2_1_and_sgen_112_2_2">Test digest of SGEN112_2_1 and SGEN 112_2_2</h3>
+<li class="level1"><div class="li"> Add 15 µl PEI(1 µg/µl) to the mix while vortexing (PEI:DNA = 3:1)<br/>
-<div class="level3">
-<div class="table sectionedit371"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0"> 2 µl </td><td class="col1"> DNA </td>
-	</tr>
-	<tr class="row2">
-		<td class="col0"> 1 µl </td><td class="col1"> 10x FD </td>
-	</tr>
-	<tr class="row3">
-		<td class="col0"> 0.5 µl </td><td class="col1"> NotI </td>
-	</tr>
-	<tr class="row4">
-		<td class="col0"> 6.5 µl </td><td class="col1"> H2O </td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_371'> </div>
-<p>
-Digest was performed at 37°C for 1 h.<br/>
-Picture in labbook\
-</p>
 </div>
-<div class='secedit editbutton_section editbutton_370'> </div>
-<h3 class="sectionedit372" id="minipreps_of_cas9-gfp_sgrna3_and_sgrna20">Minipreps of Cas9-GFP sgRNA3 and sgRNA20</h3>
-<div class="level3">
-<p>
-more colonies for Cas9-GFP sgRNA3 and sgRNA20 were sent for sequencing.<br/>
-Overnight cultures (5 ml LLB-Amp)
-</p>
-<ul>
-<li class="level1"><div class="li"> for electroporation lenti-Cas9-puro sequenced sgRNAs1-9</div>
 </li>
-<li class="level1"><div class="li"> for cloning constitutively active (CCa) HIF1A as western blot positive contro, 1x HA-HIF1A from plate</div>
+<li class="level1"><div class="li"> incubate 15 min at RT<br/>
+</div>
 </li>
-</ul>
+<li class="level1"><div class="li"> Add the mix to the culture<br/>
+</div>
+</li>
+<li class="level1"><div class="li"> incubate for 24 h<br/>
+</div>
+</li>
+</ol>
 </div>
-<div class='secedit editbutton_section editbutton_372'> </div>
+<div class='secedit editbutton_section editbutton_115'> </div>
-<h2 class="sectionedit373" id="section070817">07.08.17</h2>
+<h2 class="sectionedit117" id="section180917">18.09.17</h2>
 <div class="level2">
 </div>
-<div class='secedit editbutton_section editbutton_373'> </div>
+<div class='secedit editbutton_section editbutton_117'> </div>
-<h3 class="sectionedit374" id="minipreps_of_101_and_104">Minipreps of 101 and 104</h3>
+<h3 class="sectionedit118" id="hre_induction1">HRE induction</h3>
 <div class="level3">
 <p>
-Minipreps were performed according to manufacturer&#039;s protocol.
+- induction cancelled because cells were contaminated with bacteria<br/>
-</p>
-</div>
-<div class='secedit editbutton_section editbutton_374'> </div>
-<h3 class="sectionedit375" id="positive_sequencing_result_for_cas9-gfp-sgrna9">Positive sequencing result for Cas9-GFP-sgRNA9</h3>
-<div class="level3">
-<p>
-Colony 4 of Cas9-GFP-sgRNA9 was positive. Cas9-GFP sgRNA3 and sgRNA20 were send for sequencing (3 colonies each).
 </p>
 </div>
-<div class='secedit editbutton_section editbutton_375'> </div>
+<div class='secedit editbutton_section editbutton_118'> </div>
-<h2 class="sectionedit376" id="section080817">08.08.17</h2>
+<h2 class="sectionedit119" id="section190917">19.09.17</h2>
 <div class="level2">
 </div>
-<div class='secedit editbutton_section editbutton_376'> </div>
+<div class='secedit editbutton_section editbutton_119'> </div>
-<h3 class="sectionedit377" id="miniprep_of_ko-kit">Miniprep of KO-Kit</h3>
+<h3 class="sectionedit120" id="overnights_of_overnights_hre4x_cre4x">overnights of overnights (HRE4x, CRE4x)</h3>
 <div class="level3">
-<p>
+</div>
-Miniprep of plasmids 101, 104, 203 was performed according to manufacturer&#039;s protocol.
+<div class='secedit editbutton_section editbutton_120'> </div>
-</p>
+<h2 class="sectionedit121" id="section200917">20.09.17</h2>
+<div class="level2">
 </div>
-<div class='secedit editbutton_section editbutton_377'> </div>
+<div class='secedit editbutton_section editbutton_121'> </div>
-<h3 class="sectionedit378" id="positive_sequencing_and_picking_colonies">Positive sequencing and picking colonies</h3>
+<h3 class="sectionedit122" id="midiprep_of_cultures_from_overnights">Midiprep of cultures from overnights</h3>
 <div class="level3">
-<p>
-Sequencing was positive for Cas9-GFP sgRNa3 (colony 5). 4 new colonies of Cas9-GFP sgRNA20 were picked (LB-Amp 5ml overnight cultures).
-Cultures of Cas9-GFP sgRNA3, sgRNA9 for preps for electroporation and SGEN shRNA112 for lentisviral transduction.
-</p>
 </div>
-<div class='secedit editbutton_section editbutton_378'> </div>
+<div class='secedit editbutton_section editbutton_122'> </div>
-<h3 class="sectionedit379" id="t7e1_assay2">T7E1 assay</h3>
+<h3 class="sectionedit123" id="pei_transfection">PEI transfection</h3>
 <div class="level3">
 <p>
-T7E1 assay was performed.
+used cells: HEK cells<br/>
-</p>
-<ul>
-<li class="level1"><div class="li"> PCR 4x HEK293T CCR5delta32 positive control </div>
-    <br>
-    <img src="https://static.igem.org/mediawiki/2017/4/47/T%E2%80%93FREIBURG%E2%80%93604.png" width="500px">
-</li>
-<li class="level1"><div class="li"> PCR 1x each of Jurkat HIF1A exons 5, 6, 8, 10 negative control </div>
-</li>
-<li class="level1"><div class="li"> PCR clearup from 2% agarose gel (Qiagen kit)</div>
-</li>
-<li class="level1"><div class="li"> annealing at 95-85% 2°C/sec, 85-25°C 1% ramps(approx. 0.6°C/sec)</div>
-</li>
-<li class="level1"><div class="li"> assay 12 µl DNA + 1.5 µl 1xNEB2 </div>
-    <br>
-    <img src="https://static.igem.org/mediawiki/2017/3/35/T%E2%80%93FREIBURG%E2%80%93607.png" width="500px">
-</li>
-</ul>
-<p>
-Tested conditions
 </p>
-<div class="table sectionedit380"><table class="inline">
+<div class="table sectionedit124"><table class="inline">
 	<thead>
 	<tr class="row0">
-		<th class="col0"> </th><th class="col1"> + T7E1 </th><th class="col2"> </th><th class="col3"> </th><th class="col4"> </th><th class="col5"> </th><th class="col6"> </th><th class="col7"> </th><th class="col8"> </th><th class="col9">no T7E1</th><th class="col10"> </th>
+		<th class="col0">Plasmid number</th><th class="col1">component</th>
 	</tr>
 	</thead>
 	<tr class="row1">
-		<td class="col0"> </td><td class="col1"> pos. ctrl. </td><td class="col2"> pos. ctrl. </td><td class="col3"> pos. ctrl. </td><td class="col4"> pos. ctrl. </td><td class="col5"> pos. ctrl. </td><td class="col6"> pos. ctrl. </td><td class="col7"> pos. ctrl. </td><td class="col8"> neg. ctrl. </td><td class="col9"> pos. ctrl. </td><td class="col10"> neg. ctrl. </td>
+		<td class="col0 rightalign">  + TDAG8</td><td class="col1">CRE(4x)-GFP-CMV-mCherry + TDAG8</td>
 	</tr>
 	<tr class="row2">
-		<td class="col0"> Method </td><td class="col1"> Cycler </td><td class="col2"> Cycler </td><td class="col3"> Block </td><td class="col4"> Water </td><td class="col5"> Cycler </td><td class="col6"> Cycler </td><td class="col7"> Cycler </td><td class="col8"> Cycler </td><td class="col9"> room temp </td><td class="col10"> room temp.</td>
+		<td class="col0"> </td><td class="col1">HRE(4x)-GFP-CMV-mCherry</td>
 	</tr>
 	<tr class="row3">
-		<td class="col0"> Amount DNA </td><td class="col1"> 200 </td><td class="col2"> 200 </td><td class="col3"> 200 </td><td class="col4"> 200 </td><td class="col5"> 200 </td><td class="col6"> 300 </td><td class="col7"> 400 </td><td class="col8"> 200 </td><td class="col9"> 200 </td><td class="col10"> 200 </td>
+		<td class="col0">pIG17_008 + pIF17_009</td><td class="col1">CMV-GFP + CMV-mCherry</td>
-	</tr>
-	<tr class="row4">
-		<td class="col0"> Duration </td><td class="col1"> 10 </td><td class="col2"> 15 </td><td class="col3"> 20 </td><td class="col4"> 20 </td><td class="col5"> 20 </td><td class="col6"> 20 </td><td class="col7"> 20 </td><td class="col8"> 20 </td><td class="col9"> 20 </td><td class="col10"> 20 </td>
 	</tr>
 </table></div>
-<div class='secedit editbutton_table editbutton_380'> </div>
+<div class='secedit editbutton_table editbutton_124'> </div><ol>
+<li class="level1"><div class="li"> Split the cells 1:2, transfer into 6-well-plate (on 16.09.17)<br/>
 </div>
-<div class='secedit editbutton_section editbutton_379'> </div>
+</li>
-<h2 class="sectionedit381" id="ko-confirm_pcr_with_primers_228_and_229">KO-Confirm PCR with primers 228 and 229</h2>
+<li class="level1"><div class="li"> Thaw PEI reagent at RT<br/>
-<div class="level2">
+</div>
+</li>
+<li class="level1"><div class="li"> Dilute plasmid in 1 ml serum free DMEM (2.5 µg from each plasmid)<br/>
+</div>
+</li>
+<li class="level1"><div class="li"> Add 15 µl PEI(1 µg/µl) to the mix while vortexing (PEI:DNA = 3:1)<br/>
+</div>
+</li>
+<li class="level1"><div class="li"> incubate 15 min at RT<br/>
+</div>
+</li>
+<li class="level1"><div class="li"> Add the mix to the culture<br/>
+</div>
+</li>
+<li class="level1"><div class="li"> incubate for 24 h<br/>
+</div>
+</li>
+</ol>
-<p>
+</div>
-PCR was performed on plasmid 104 and gDNA to confirm knockout.<br/>
+<div class='secedit editbutton_section editbutton_123'> </div>
+<h3 class="sectionedit125" id="filtration_of_dmem_addition_of_fbs">Filtration of DMEM, addition of FBS</h3>
+<div class="level3">
-</p>
-<div class="table sectionedit382"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0"> 2 µl </td><td class="col1"> template </td>
-	</tr>
-	<tr class="row2">
-		<td class="col0"> 1.5 µl </td><td class="col1"> 10x buffer </td>
-	</tr>
-	<tr class="row3">
-		<td class="col0"> 0.3 µl </td><td class="col1"> dNTPs </td>
-	</tr>
-	<tr class="row4">
-		<td class="col0"> 0.75 µl </td><td class="col1"> primer fw </td>
-	</tr>
-	<tr class="row5">
-		<td class="col0"> 0.75 µl </td><td class="col1"> primer rv </td>
-	</tr>
-	<tr class="row6">
-		<td class="col0"> 0.2 µl </td><td class="col1"> Taq </td>
-	</tr>
-	<tr class="row7">
-		<td class="col0"> 9.5 µl </td><td class="col1"> H2O </td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_382'> </div>
 <p>
-Program koconfirm (but here at 64°C)<br/>
+- filter DMEM sterile<br/>
-</p>
+- add 2 % FBS<br/>
-<div class="table sectionedit383"><table class="inline">
-	<thead>
+- set up pH of aliquots with HCl (4 M)<br/>
-	<tr class="row0">
-		<th class="col0">step</th><th class="col1">temperature (°C)</th><th class="col2">time</th>
+- final pH values: 6.7, 7.1, 7.4<br/>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0"> 1 </td><td class="col1"> 95 </td><td class="col2"> 3:00 </td>
-	</tr>
-	<tr class="row2">
-		<td class="col0"> 2 </td><td class="col1"> 95 </td><td class="col2"> 0:30 </td>
-	</tr>
-	<tr class="row3">
-		<td class="col0"> 3 </td><td class="col1"> 59 </td><td class="col2"> 0:30 </td>
-	</tr>
-	<tr class="row4">
-		<td class="col0"> 4 </td><td class="col1"> 72 </td><td class="col2"> 0:40 </td>
-	</tr>
-	<tr class="row5">
-		<td class="col0"> 5 </td><td class="col1"> 72 </td><td class="col2"> 7:00 </td>
-	</tr>
-	<tr class="row6">
-		<td class="col0"> 6 </td><td class="col1"> 4 </td><td class="col2"> pause </td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_383'> </div>
-<p>
-Steps 2 to 4 were repeated 30x.
-</p>
-<p>
-Picture no. 614 lower line
 </p>
 </div>
-<div class='secedit editbutton_section editbutton_381'> </div>
+<div class='secedit editbutton_section editbutton_125'> </div>
-<h3 class="sectionedit384" id="t7e1_assay_for_ccr5delta32_positive_control">T7E1 assay for CCR5delta32 positive control</h3>
+<h2 class="sectionedit126" id="section220917">22.09.17</h2>
-<div class="level3">
-<div class="table sectionedit385"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0"> 5.5 µl </td><td class="col1"> CCR5delta32 DNA (209 ng) </td>
-	</tr>
-	<tr class="row2">
-		<td class="col0"> 12.5 µl </td><td class="col1"> H2O </td>
-	</tr>
-	<tr class="row3">
-		<td class="col0"> 2 µl </td><td class="col1"> NEB10x </td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_385'> </div>
-</div>
-<div class='secedit editbutton_section editbutton_384'> </div>
-<h2 class="sectionedit386" id="section100817">10.08.17</h2>
 <div class="level2">
 </div>
-<div class='secedit editbutton_section editbutton_386'> </div>
+<div class='secedit editbutton_section editbutton_126'> </div>
-<h3 class="sectionedit387" id="pcr_for_t7_assay_annealing_and_t7_incubation">PCR for T7 assay, annealing and T7 incubation</h3>
+<h3 class="sectionedit127" id="induction_of_cre_and_hre_in_hek_cells">induction of CRE and HRE in HEK cells</h3>
 <div class="level3">
 <p>
-Experiment failed.
+- indution in 96-wel-plates
-</p>
+- cells transiently transfected with CRE-GFP-CMV-mCherry, add acid<br/>
-</div>
+- cells transiently transfected with HRE-GFP-CMV-mCherry, add CoCl2<br/>
-<div class='secedit editbutton_section editbutton_387'> </div>
-<h3 class="sectionedit388" id="mycoplasm_pcr">Mycoplasm PCR</h3>
-<div class="level3">
-<div class="table sectionedit389"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">volume</th><th class="col2">component</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0"> 2 µl </td><td class="col1"> </td><td class="col2"> template </td>
-	</tr>
-	<tr class="row2">
-		<td class="col0"> 1.5 µl </td><td class="col1"> 13.5 µl </td><td class="col2"> 10x Y Buffer </td>
-	</tr>
-	<tr class="row3">
-		<td class="col0"> 0.3 µl </td><td class="col1"> 2.7 µl </td><td class="col2"> dNTPs </td>
-	</tr>
-	<tr class="row4">
-		<td class="col0"> 1 µl </td><td class="col1"> 9 µl </td><td class="col2"> primer fw </td>
-	</tr>
-	<tr class="row5">
-		<td class="col0"> 1 µl </td><td class="col1"> 9 µl </td><td class="col2"> primer rv </td>
-	</tr>
-	<tr class="row6">
-		<td class="col0"> 0.2 µl </td><td class="col1"> 1.8 µl </td><td class="col2"> Taq </td>
-	</tr>
-	<tr class="row7">
-		<td class="col0"> 9 µl </td><td class="col1"> 81 µl </td><td class="col2"> H2O </td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_389'> </div>
-<p>
-Program: myco<br/>
-</p>
+- plate reader: no measurable induction of GFP<br/>
-<div class="table sectionedit390"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">step</th><th class="col1">temperature (°C)</th><th class="col2">time</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0"> 1 </td><td class="col1"> 95 </td><td class="col2"> 5:00 </td>
-	</tr>
-	<tr class="row2">
-		<td class="col0"> 2 </td><td class="col1"> 95 </td><td class="col2"> 0:40 </td>
-	</tr>
-	<tr class="row3">
-		<td class="col0"> 3 </td><td class="col1"> 60 </td><td class="col2"> 0:40 </td>
-	</tr>
-	<tr class="row4">
-		<td class="col0"> 4 </td><td class="col1"> 72 </td><td class="col2"> 0:40 </td>
-	</tr>
-	<tr class="row5">
-		<td class="col0"> 5 </td><td class="col1"> 72 </td><td class="col2"> 5:00 </td>
-	</tr>
-	<tr class="row6">
-		<td class="col0"> 6 </td><td class="col1"> 4 </td><td class="col2"> pause </td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_390'> </div>
-<p>
-Steps 2 to 4 were repeated 30x.
-</p>
-<p>
-Picture no. 621
 </p>
 </div>
-<div class='secedit editbutton_section editbutton_388'> </div>
+<div class='secedit editbutton_section editbutton_127'> </div>
-<h2 class="sectionedit391" id="section110817">11.08.17</h2>
+<h2 class="sectionedit128" id="section230917">23.09.17</h2>
 <div class="level2">
 </div>
-<div class='secedit editbutton_section editbutton_391'> </div>
+<div class='secedit editbutton_section editbutton_128'> </div>
-<h3 class="sectionedit392" id="ko-confirm_primer_228_and_229">KO-confirm primer 228 and 229</h3>
+<h3 class="sectionedit129" id="facs_analysis_of_hek_cells">FACS analysis of HEK cells</h3>
 <div class="level3">
 <p>
-To confirm knockout a PCR was performed.
+- FACS analysis after CRE induction and HRE induction<br/>
-</p>
-<div class="table sectionedit393"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">volume</th><th class="col2">component</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0"> 2 µl </td><td class="col1"> </td><td class="col2"> template </td>
-	</tr>
-	<tr class="row2">
-		<td class="col0"> 1.5 µl </td><td class="col1"> 7.5 µl </td><td class="col2"> 10x buffer </td>
-	</tr>
-	<tr class="row3">
-		<td class="col0"> 0.5 µl </td><td class="col1"> 2.5 µl </td><td class="col2"> dNTPs </td>
-	</tr>
-	<tr class="row4">
-		<td class="col0"> 0.75 µl </td><td class="col1"> 3.75 µl </td><td class="col2"> primer fw </td>
-	</tr>
-	<tr class="row5">
-		<td class="col0"> 0.75 µl </td><td class="col1"> 3.75 µl </td><td class="col2"> primer rv </td>
-	</tr>
-	<tr class="row6">
-		<td class="col0"> 0.2 µl </td><td class="col1 leftalign">  </td><td class="col2"> Taq </td>
-	</tr>
-	<tr class="row7">
-		<td class="col0"> 9.3 µl </td><td class="col1"> 46.5 µl </td><td class="col2"> H2O </td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_393'> </div>
-</div>
-<div class='secedit editbutton_section editbutton_392'> </div>
-<h3 class="sectionedit394" id="pcr_for_t7e1_assay1">PCR for T7E1 assay</h3>
-<div class="level3">
-<p>
-PCR was performed as previously described (double volume 50 µl).<br/>
 </p>
-<div class="table sectionedit395"><table class="inline">
+<div class="table sectionedit130"><table class="inline">
 	<thead>
 	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
+		<th class="col0">CREx4_GFP</th><th class="col1">HREx4_GFP</th>
 	</tr>
 	</thead>
 	<tr class="row1">
-		<td class="col0">1 µl </td><td class="col1"> gDNA </td>
+		<td class="col0"><img style="max-height: 300px" src="https://static.igem.org/mediawiki/2017/3/34/T-Freiburg-9_23_2017_facs_analysis_1.png"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=cre_67_71_75.png" class="media" title="cre_67_71_75.png"><img src="/igem2017/lib/exe/fetch.php?w=200&amp;tok=06e2a0&amp;media=cre_67_71_75.png" class="mediacenter" alt="" width="200" /></a></td><td class="col1"><img style="max-height: 300px" src="https://static.igem.org/mediawiki/2017/9/94/T-Freiburg-9_23_2017_facs_analysis_2.png"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=hre_facs.png" class="media" title="hre_facs.png"><img src="/igem2017/lib/exe/fetch.php?w=200&amp;tok=d7532c&amp;media=hre_facs.png" class="mediacenter" alt="" width="200" /></a></td>
 	</tr>
 	<tr class="row2">
-		<td class="col0"> 10 µl </td><td class="col1"> 5x buffer </td>
+		<td class="col0"><img style="max-height: 300px" src="https://static.igem.org/mediawiki/2017/f/f2/T-Freiburg-9_23_2017_facs_analysis_3.png"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=cre_ph_67.png" class="media" title="cre_ph_67.png"><img src="/igem2017/lib/exe/fetch.php?w=200&amp;tok=3baddb&amp;media=cre_ph_67.png" class="mediacenter" alt="" width="200" /></a></td><td class="col1"> </td>
-	</tr>
-	<tr class="row3">
-		<td class="col0"> 1.5 µl </td><td class="col1"> dNTPs (10µM) </td>
-	</tr>
-	<tr class="row4">
-		<td class="col0"> 1.25 µl </td><td class="col1"> primer fw </td>
-	</tr>
-	<tr class="row5">
-		<td class="col0"> 1.25 µl </td><td class="col1"> primer rv </td>
-	</tr>
-	<tr class="row6">
-		<td class="col0"> 0.5 µl </td><td class="col1"> phusion </td>
 	</tr>
 </table></div>
-<div class='secedit editbutton_table editbutton_395'> </div>
+<div class='secedit editbutton_table editbutton_130'> </div>
 </div>
-<div class='secedit editbutton_section editbutton_394'> </div>
+<div class='secedit editbutton_section editbutton_129'> </div>
-<h3 class="sectionedit396" id="test_digest_hif_pcdna3">Test digest HIF pcDNA3</h3>
+<h2 class="sectionedit131" id="section240917">24.09.17</h2>
-<div class="level3">
+<div class="level2">
-<div class="table sectionedit397"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0"> 5 µl </td><td class="col1"> DNA HIF pcDNA3 </td>
-	</tr>
-	<tr class="row2">
-		<td class="col0"> 1 µl </td><td class="col1"> CutSmart buffer </td>
-	</tr>
-	<tr class="row3">
-		<td class="col0"> 1 µl </td><td class="col1"> XhoI </td>
-	</tr>
-	<tr class="row4">
-		<td class="col0"> 3 µl </td><td class="col1"> H2O </td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_397'> </div>
-<p>
-Test digest was performed for 1 hour at 37°C (was accidentally thrown away).
-</p>
 </div>
-<div class='secedit editbutton_section editbutton_396'> </div>
+<div class='secedit editbutton_section editbutton_131'> </div>
-<h3 class="sectionedit398" id="test_digest_of_lenti-cas9-sgrna20_and_knockdown_plamsids">Test digest of lenti-Cas9-sgRNA20 and knockdown plamsids</h3>
+<h3 class="sectionedit132" id="repetition_of_the_interlab_study1">Repetition of the Interlab Study</h3>
 <div class="level3">
 <p>
-lenti-Cas9-sgRNA20 and knockdown SGEN112_2.1 and SGEN112_2.2 were test digested.<br/>
+- transformation of DH5-α with plasmids from Kit 6 (20B, 20D, 20F, 20H, 20L, 20N, 20P)<br/>
-</p>
+- incubation at 37 °C<br/>
-<div class="table sectionedit399"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0"> 1 µl </td><td class="col1"> BamHI </td>
-	</tr>
-	<tr class="row2">
-		<td class="col0"> 2 µl </td><td class="col1"> Plasmid </td>
-	</tr>
-	<tr class="row3">
-		<td class="col0"> 1.5 µl </td><td class="col1"> FD buffer </td>
-	</tr>
-	<tr class="row4">
-		<td class="col0"> 10.5 µl </td><td class="col1"> H2O </td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_399'> </div>
-<p>
-Lenti-Cas9<br/>
-</p>
-<div class="table sectionedit400"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0"> 1 µl </td><td class="col1"> NcoI </td>
-	</tr>
-	<tr class="row2">
-		<td class="col0"> 2 µl </td><td class="col1"> plasmid </td>
-	</tr>
-	<tr class="row3">
-		<td class="col0"> 1.5 µl </td><td class="col1"> FD buffer </td>
-	</tr>
-	<tr class="row4">
-		<td class="col0"> 10.5 µl </td><td class="col1"> H2O </td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_400'> </div>
-</div>
-<div class='secedit editbutton_section editbutton_398'> </div>
-<h3 class="sectionedit401" id="t7e1_assay3">T7E1 assay</h3>
-<div class="level3">
-<p>
-µl PCR samples (from 11.08.17) were annealed for longer than 3 h in heatblock, then 10 min at 95°C and cooled down in block. 400 ng/sample PCR product were obtained (reaction mix as previously; time: 18, 20, 22 min T7E1 at 37°C, stop with 3 µl 6x Orange Loading Dy on ice. Jurkat HIF1A exon 5 from PCR on 08.08.17 served as negative control and was incubated for 20 min with T7. 2% EtBR gel was used. Expected bands at 200 bp and 300 bp were observed in all three CCR5delta32 samples with T7.
 </p>
 </div>
-<div class='secedit editbutton_section editbutton_401'> </div>
+<div class='secedit editbutton_section editbutton_131'> </div>
-<h2 class="sectionedit402" id="section130817">13.08.17</h2>
+<h2 class="sectionedit132" id="section290917">29.09.17</h2>
 <div class="level2">
 </div>
-<div class='secedit editbutton_section editbutton_402'> </div>
+<div class='secedit editbutton_section editbutton_132'> </div>
-<h3 class="sectionedit403" id="ko-confirm_primer_228_and_2291">KO-confirm primer 228 and 229</h3>
+<h3 class="sectionedit133" id="calibration_of_plate_reader">calibration of plate reader</h3>
 <div class="level3">
-<div class="table sectionedit404"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">volume</th><th class="col2">component</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0"> 2 µl </td><td class="col1"> </td><td class="col2"> template </td>
-	</tr>
-	<tr class="row2">
-		<td class="col0"> 1.5 µl </td><td class="col1"> 7.5 </td><td class="col2"> 10x buffer </td>
-	</tr>
-	<tr class="row3">
-		<td class="col0"> 1 µl </td><td class="col1"> 5 µl </td><td class="col2"> dNTPs </td>
-	</tr>
-	<tr class="row4">
-		<td class="col0"> 1 µl </td><td class="col1"> 5 µl </td><td class="col2"> primer fw </td>
-	</tr>
-	<tr class="row5">
-		<td class="col0"> 1 µl </td><td class="col1"> 5 µl </td><td class="col2"> primer rv </td>
-	</tr>
-	<tr class="row6">
-		<td class="col0"> 0.2 µl </td><td class="col1 leftalign">  </td><td class="col2"> Taq </td>
-	</tr>
-	<tr class="row7">
-		<td class="col0"> 8.2 µl </td><td class="col1"> 41 µl </td><td class="col2"> H2O </td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_404'> </div>
 <p>
-program: koconfirm<br/>
+- used cells: knockdown-cells + untransfected cells, compositions 1:0, 1:1, 1:2, 1:4, 0:1<br/>
-picture no. 633<br/>
+- 96-well-plates, black, clear bottom, 100 000 cells per well<br/>
+- different media: RPMI164, DMEM, DMEM and PBS (500 000 cells per well)
 </p>
-</div>
+<img style="max-height: 400px" src="https://static.igem.org/mediawiki/2017/9/9a/T-Freiburg-9_29_2017_calibration_1.jpeg">
-<div class='secedit editbutton_section editbutton_403'> </div>
-<h3 class="sectionedit405" id="retrafo_of_sgrna20_4">Retrafo of sgRNA20_4</h3>
-<div class="level3">
-<div class="table sectionedit406"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0"> 2 µl </td><td class="col1"> template (1:1 genomic:plasmid)</td>
-	</tr>
-	<tr class="row2">
-		<td class="col0"> 1.5 µl </td><td class="col1"> buffer 10x </td>
-	</tr>
-	<tr class="row3">
-		<td class="col0"> 1 µl </td><td class="col1"> dNTPs </td>
-	</tr>
-	<tr class="row4">
-		<td class="col0"> 1 µl </td><td class="col1"> primer fw </td>
-	</tr>
-	<tr class="row5">
-		<td class="col0"> 1 µl </td><td class="col1"> primer rv </td>
-	</tr>
-	<tr class="row6">
-		<td class="col0"> 0.2 µl </td><td class="col1"> Taq </td>
-	</tr>
-	<tr class="row7">
-		<td class="col0"> 8.3 µl </td><td class="col1"> H2O </td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_406'> </div>
-<p>
-Program: PCR kitKOconfirm<br/>
-</p>
-<div class="table sectionedit407"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">step</th><th class="col1">temperature (°C)</th><th class="col2">time</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0"> 1 </td><td class="col1"> 94 </td><td class="col2"> 5:00 </td>
-	</tr>
-	<tr class="row2">
-		<td class="col0"> 2 </td><td class="col1"> 94 </td><td class="col2"> 0:40 </td>
-	</tr>
-	<tr class="row3">
-		<td class="col0"> 3 </td><td class="col1"> 60 </td><td class="col2"> 0:40 </td>
-	</tr>
-	<tr class="row4">
-		<td class="col0"> 4 </td><td class="col1"> 72 </td><td class="col2"> 4:00 </td>
-	</tr>
-	<tr class="row5">
-		<td class="col0"> 5 </td><td class="col1"> 72 </td><td class="col2"> 5:00 </td>
-	</tr>
-	<tr class="row6">
-		<td class="col0"> 6 </td><td class="col1"> 4 </td><td class="col2"> pause </td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_407'> </div>
 <p>
-Steps 2 to 4 were repeated 35x.
+<a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=calibration_kd_cells.jpg" class="media" title="calibration_kd_cells.jpg"><img src="/igem2017/lib/exe/fetch.php?w=400&amp;tok=a325d9&amp;media=calibration_kd_cells.jpg" class="mediacenter" alt="" width="400" /></a>
 </p>
 </div>
-<div class='secedit editbutton_section editbutton_405'> </div>
+<div class='secedit editbutton_section editbutton_134'> </div>
-<h2 class="sectionedit408" id="section140817">14.08.17</h2>
+<h3 class="sectionedit135" id="induction_of_hre_4x">induction of HRE(4x)</h3>
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_408'> </div>
-<h3 class="sectionedit409" id="pcr_ko-kit_confirm_taq_and_q5">PCR KO-Kit confirm (TAq and Q5)</h3>
 <div class="level3">
-<p>
-A PCR was performed to confirm knockout.<br/>
-</p>
-<div class="table sectionedit410"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0"> 2 µl </td><td class="col1"> template gDNA </td>
-	</tr>
-	<tr class="row2">
-		<td class="col0"> 1.5 µl </td><td class="col1"> buffer 10x </td>
-	</tr>
-	<tr class="row3">
-		<td class="col0"> 1 µl </td><td class="col1"> dNTPs </td>
-	</tr>
-	<tr class="row4">
-		<td class="col0"> 1 µl </td><td class="col1"> primer fw 32 </td>
-	</tr>
-	<tr class="row5">
-		<td class="col0"> 1 µl </td><td class="col1"> primer rv 60 </td>
-	</tr>
-	<tr class="row6">
-		<td class="col0"> 0.5 µl </td><td class="col1"> Taq Q5 </td>
-	</tr>
-	<tr class="row7">
-		<td class="col0"> 7.5 µl </td><td class="col1"> H2O </td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_410'> </div>
 <p>
-Program: kokit3primers<br/>
+- used cells: HEK stably transfected with HRE(4x)-GFP-CMV-mCherry, untransfected as a negative control<br/>
-</p>
+- induction in 24-well-plate<br/>
-<div class="table sectionedit411"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">step</th><th class="col1">temperature (°C)</th><th class="col2">time</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0"> 1 </td><td class="col1"> 95.0 </td><td class="col2"> 2:00 </td>
-	</tr>
-	<tr class="row2">
-		<td class="col0"> 2 </td><td class="col1"> 95.0 </td><td class="col2"> 0:30 </td>
-	</tr>
-	<tr class="row3">
-		<td class="col0"> 3 </td><td class="col1"> 55.5 </td><td class="col2"> 0:40 </td>
-	</tr>
-	<tr class="row4">
-		<td class="col0"> 4 </td><td class="col1"> 72.0 </td><td class="col2"> 2:00 </td>
-	</tr>
-	<tr class="row5">
-		<td class="col0"> 5 </td><td class="col1"> 72.0 </td><td class="col2"> 5:00 </td>
-	</tr>
-	<tr class="row6">
-		<td class="col0"> 6 </td><td class="col1"> 4.0 </td><td class="col2"> pause </td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_411'> </div>
-<p>
-Steps 2 to 4 were repeated 35x.
-</p>
-<p>
+- add CoCl2: 0 µM, 20 µM, 40 µM, 80 µM, 100 µM, 320 µM, 640 µM<br/>
-Product ~1.6 kb at 54.5°C, ET 1.5 min<br/>
-Picture no. 642<br/>
+- FACS analysis<br/>
+<img style="max-height: 400px" src="https://static.igem.org/mediawiki/2017/4/42/T-Freiburg-9_29_2017_induction_of_HRE_1.jpeg">
 </p>
 </div>
-<div class='secedit editbutton_section editbutton_409'> </div>
+</div>
-<h3 class="sectionedit412" id="gibson_hif1a">Gibson HIF1A</h3>
+ <button class="accordion">October</button>
-<div class="level3">
+         <div class="panel panel-default">
-<ul>
-<li class="level1"><div class="li"> Insert HIF1A 20 ng/µl, 520 bp</div>
-</li>
-<li class="level1"><div class="li"> Backbone pcDNA3 8 ng/µl, 7464 bp</div>
-</li>
-<li class="level1"><div class="li"> Insert:backbone 4:1</div>
-</li>
-</ul>
-<div class="table sectionedit413"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0"> 10 µl </td><td class="col1"> 2x gibson </td>
-	</tr>
-	<tr class="row2">
-		<td class="col0"> 9 µl </td><td class="col1"> backbone </td>
-	</tr>
-	<tr class="row3">
-		<td class="col0"> 1 µl </td><td class="col1"> insert </td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_413'> </div>
-<p>
-Assembly was performed for 1 h at 50°C.<br/>
-</p>
+<div class='secedit editbutton_section editbutton_134'> </div>
+<h2 class="sectionedit135" id="section021017">02.10.17</h2>
-<p>
-XL10gold were transformed using heat shock protocol (06.05.17).
-</p>
-</div>
-<div class='secedit editbutton_section editbutton_412'> </div>
-<h2 class="sectionedit414" id="section150817">15.08.17</h2>
 <div class="level2">
 </div>
-<div class='secedit editbutton_section editbutton_414'> </div>
+<div class='secedit editbutton_section editbutton_135'> </div>
-<h3 class="sectionedit415" id="pcr_ko-kit_confirm_3_primers">PCR KO-Kit confirm 3 primers</h3>
+<h3 class="sectionedit136" id="ph_measurements_with_hek_cells">pH measurements with HEK cells</h3>
 <div class="level3">
 <p>
-A PCR was performed to confirm knockout.
+- 6-well-plate, 900 000 cells per well (90% confluency)<br/>
-</p>
-<div class="table sectionedit416"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0"> 2 µl </td><td class="col1"> template gDNA </td>
-	</tr>
-	<tr class="row2">
-		<td class="col0"> 1.5 µl </td><td class="col1"> buffer 10x </td>
-	</tr>
-	<tr class="row3">
-		<td class="col0"> 1 µl </td><td class="col1"> dNTPs </td>
-	</tr>
-	<tr class="row4">
-		<td class="col0"> 1 µl </td><td class="col1"> primer fw 32 </td>
-	</tr>
-	<tr class="row5">
-		<td class="col0"> 1 µl </td><td class="col1"> primer rv 60 </td>
-	</tr>
-	<tr class="row6">
-		<td class="col0"> 0.3 µl </td><td class="col1"> Taq (Q5) </td>
-	</tr>
-	<tr class="row7">
-		<td class="col0"> 7.7 µl </td><td class="col1"> H2O </td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_416'> </div>
-<p>
-Program: kokit confirm 3 primers<br/>
-at 34,5°C, elongation time 1.5 min<br/>
+- DMEM (5 ml per well), pH set to 6.8<br/>
-picture no. 648
-</p>
-<div class="table sectionedit417"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0"> 2 µl </td><td class="col1"> template gDNA </td>
-	</tr>
-	<tr class="row2">
-		<td class="col0"> 1.5 µl </td><td class="col1"> buffer 10x </td>
-	</tr>
-	<tr class="row3">
-		<td class="col0"> 1 µl </td><td class="col1"> dNTPs </td>
-	</tr>
-	<tr class="row4">
-		<td class="col0"> 1 µl </td><td class="col1"> primer fw 32 </td>
-	</tr>
-	<tr class="row5">
-		<td class="col0"> 1 µl </td><td class="col1"> primer rv 60 </td>
-	</tr>
-	<tr class="row6">
-		<td class="col0"> 0.5 µl </td><td class="col1"> Taq (Q5) </td>
-	</tr>
-	<tr class="row7">
-		<td class="col0"> 7.5 µl </td><td class="col1"> H2O </td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_417'> </div>
-<p>
-Program: kokit confirm 3 primers<br/>
-at 49°C, elongation time 2 min<br/>
+- after 6 h: pH=7.2<br/>
-Picture no. 648
 </p>
 </div>
-<div class='secedit editbutton_section editbutton_415'> </div>
+<div class='secedit editbutton_section editbutton_136'> </div>
-<h3 class="sectionedit418" id="miniprep_of_lenti-cas_sgrna20_4_and_test_digest_with_ecori_and_bamhi">Miniprep of Lenti-Cas sgRNA20_4 and test digest with EcoRI and BamHI</h3>
+<h3 class="sectionedit137" id="induction_of_hre">induction of HRE</h3>
 <div class="level3">
 <p>
-Miniprep was performed according to manufacturer&#039;s protocol.
+- used cells: Jurkat and HEK, stably transfected with HRE(4x)-GFP-CMV-mCherry<br/>
+- induction with CoCl2: 0 µM, 50 µM, 100 µM, 150 µM, 200 µM, 250 µM, 300 µM, 350 µM<br/>
 </p>
-<div class="table sectionedit419"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0"> 2 µl </td><td class="col1"> DNA </td>
-	</tr>
-	<tr class="row2">
-		<td class="col0"> 0.75 µl </td><td class="col1"> EcoRI </td>
-	</tr>
-	<tr class="row3">
-		<td class="col0"> 0.75 µl </td><td class="col1"> BamHI </td>
-	</tr>
-	<tr class="row4">
-		<td class="col0"> 1.5 µl </td><td class="col1"> 10x FD </td>
-	</tr>
-	<tr class="row5">
-		<td class="col0"> 5 µl </td><td class="col1"> H2O </td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_419'> </div>
 </div>
-<div class='secedit editbutton_section editbutton_418'> </div>
+<div class='secedit editbutton_section editbutton_137'> </div>
-<h3 class="sectionedit420" id="pcr_cahif1a">PCR CaHIF1A</h3>
+<h3 class="sectionedit138" id="ph_measurements_with_hek_cells1">pH measurements with HEK cells</h3>
 <div class="level3">
 <p>
-Protocol 13.08.17<br/>
+- 6-well-plate, 900 000 cells per well<br/>
+- DMEM (5 ml per well), pH set to 6.73<br/>
-only backbone, 1 µl dNTPs instead
 </p>
 </div>
-<div class='secedit editbutton_section editbutton_420'> </div>
+<div class='secedit editbutton_section editbutton_138'> </div>
-<h2 class="sectionedit421" id="section160817">16.08.17</h2>
+<h2 class="sectionedit139" id="section031017">03.10.17</h2>
 <div class="level2">
 </div>
-<div class='secedit editbutton_section editbutton_421'> </div>
+<div class='secedit editbutton_section editbutton_139'> </div>
-<h3 class="sectionedit422" id="pcr_ko-kit">PCR KO-Kit</h3>
+<h3 class="sectionedit140" id="indcution_of_cre_4x_in_hek_cells">indcution of CRE(4x) in HEK cells</h3>
 <div class="level3">
 <p>
-gDNA101 template was diluted 1:10 diluted.<br/>
+- HEK stably transfected with CRE-GFP-CMV-mCherry, induction with acid (pH 6.7, 7.4, 8.1)<br/>
-</p>
+- FACS measurement failed<br/>
-<p>
-Primers 228 and 229<br/>
-</p>
-<div class="table sectionedit423"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0"> 1 µl </td><td class="col1"> template </td>
-	</tr>
-	<tr class="row2">
-		<td class="col0"> 2 µl </td><td class="col1"> buffer </td>
-	</tr>
-	<tr class="row3">
-		<td class="col0"> 1 µl </td><td class="col1"> primer fw </td>
-	</tr>
-	<tr class="row4">
-		<td class="col0"> 1 µl </td><td class="col1"> primer rev </td>
-	</tr>
-	<tr class="row5">
-		<td class="col0"> 1 µl </td><td class="col1"> dNTPs </td>
-	</tr>
-	<tr class="row6">
-		<td class="col0"> 0.5 µl </td><td class="col1"> Taq (Q5) </td>
-	</tr>
-	<tr class="row7">
-		<td class="col0"> 13.5 </td><td class="col1"> H2O </td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_423'> </div>
-<p>
-Primers 32 and 60
-</p>
-<div class="table sectionedit424"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0"> 1 µl </td><td class="col1"> template </td>
-	</tr>
-	<tr class="row2">
-		<td class="col0"> 2 µl </td><td class="col1"> buffer </td>
-	</tr>
-	<tr class="row3">
-		<td class="col0"> 1 µl </td><td class="col1"> primer fw </td>
-	</tr>
-	<tr class="row4">
-		<td class="col0"> 1 µl </td><td class="col1"> primer rev </td>
-	</tr>
-	<tr class="row5">
-		<td class="col0"> 1 µl </td><td class="col1"> dNTPs </td>
-	</tr>
-	<tr class="row6">
-		<td class="col0"> 0.5 µl </td><td class="col1"> Taq (Q5) </td>
-	</tr>
-	<tr class="row7">
-		<td class="col0"> 13.5 </td><td class="col1"> H2O </td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_424'> </div>
-<p>
-Picture no. 654<br/>
 </p>
 </div>
-<div class='secedit editbutton_section editbutton_422'> </div>
+<div class='secedit editbutton_section editbutton_141'> </div>
-<h3 class="sectionedit425" id="pcr_on_cahif1a_gel_gelex_gibson_and_trafo">PCR on CaHIF1a gel, gelex, gibson and trafo</h3>
+<h3 class="sectionedit142" id="facs_analysis_of_cocl2_treated_hek_cells">FACS analysis of CoCl2 treated HEK cells</h3>
 <div class="level3">
-<p>
-Performed as described previously.
-</p>
 </div>
-<div class='secedit editbutton_section editbutton_425'> </div>
+<div class='secedit editbutton_section editbutton_142'> </div>
-<h3 class="sectionedit426" id="preparation_of_buffers_for_electrophoresis">Preparation of buffers for electrophoresis</h3>
+<h2 class="sectionedit143" id="section051017">05.10.17</h2>
-<div class="level3">
+<div class="level2">
 <ul>
-<li class="level1"><div class="li"> Stacking gel stock: Tris-HCL 1 M pH 6.8</div>
+<li class="level1"><div class="li"> resuspended HEK cells containing stable CREx4 were diluted at different ratios with wildtype HEK cells were incubated at different pH values (6.5, 7.1, 7.7) and measured on a plate reader.</div>
 </li>
-<li class="level1"><div class="li"> Separation gel stock: Tris-HCL 1.5 M pH 8.8</div>
+<li class="level1"><div class="li"> normalization via OD600</div>
-</li>
-<li class="level1"><div class="li"> WB running buffer 10x: Tris 0.5 M, 0.1% SDS, glycin 1 M, HCl –&gt; pH 8.3</div>
-</li>
-<li class="level1"><div class="li"> 10x Lämmli running buffer (1 l): 30 g tris, 10 g SDS, 144 g glycin</div>
 </li>
 </ul>
-</div>
+<img style="max-height: 400px" src="https://static.igem.org/mediawiki/2017/c/c2/T-Freiburg-10_5_2017_plate_reader_1.jpeg">
-<div class='secedit editbutton_section editbutton_426'> </div>
-<h3 class="sectionedit427" id="protein_extraction_from_hek">Protein extraction from HEK</h3>
-<div class="level3">
 <p>
-HRE-SEAP and CoCl2 treated cells were lysed to extract proteins (12 well plate triplicates, WT, 0µM / 200 µM / 500 µM CoCl2).
+<a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=platereader_result.jpg" class="media" title="platereader_result.jpg"><img src="/igem2017/lib/exe/fetch.php?w=400&amp;tok=786c89&amp;media=platereader_result.jpg" class="mediacenter" alt="" width="400" /></a>
 </p>
-<p>
-Protocol cell lysis
-</p>
-<ul>
-<li class="level1"><div class="li"> on ice 2x washing in PBS (remove by 2 min, 100xg) with 100µM CoCl2 for CoCl2 cells</div>
-</li>
-<li class="level1"><div class="li"> 150 µl RIPA buffer, 0.1% proteinase inhibitor cocktail with 100µM CoCl2 for CoCl2 cells</div>
-</li>
-<li class="level1"><div class="li"> 5 min ultrasound</div>
-</li>
-<li class="level1"><div class="li"> 20 min 20,000xg, 4°C –&gt; take supernatant (pellet stored for control)</div>
-</li>
-</ul>
 </div>
-<div class='secedit editbutton_section editbutton_427'> </div>
+<div class='secedit editbutton_section editbutton_143'> </div>
-<h3 class="sectionedit428" id="sds-page">SDS-PAGE</h3>
+<h2 class="sectionedit144" id="section141017">14.10.17</h2>
-<div class="level3">
+<div class="level2">
-<p>
-Protocol SDS-PAGE
-</p>
-<ul>
-<li class="level1"><div class="li"> 8% acrylamid gel</div>
-</li>
-<li class="level1"><div class="li"> 4.5% stacking gel</div>
-</li>
-<li class="level1"><div class="li"> heat samples 10 min 96°C in loading dye</div>
-</li>
-<li class="level1"><div class="li"> 1x Lämmli running buffer</div>
-</li>
-<li class="level1"><div class="li"> run at 80 V until end of stacking gel</div>
-</li>
-<li class="level1"><div class="li"> 150 V for rest of run (until blue loading dye elutes)</div>
-</li>
-</ul>
 </div>
-<div class='secedit editbutton_section editbutton_428'> </div>
+<div class='secedit editbutton_section editbutton_144'> </div>
-<h3 class="sectionedit429" id="western_blot">Western Blot</h3>
+<h3 class="sectionedit145" id="repetition_of_the_interlab_study2">Repetition of the Interlab Study</h3>
 <div class="level3">
 <p>
-Protocol Western Blot
+- transformation of positive control, negative control, Device 1-6
 </p>
-<ul>
-<li class="level1"><div class="li"> assemble in running buffer</div>
-</li>
-<li class="level1"><div class="li"> incubate/soak gel, whatman papers, sponges in running buffer</div>
-</li>
-<li class="level1"><div class="li"> activate PVDF membrane in 100% methanol for 1 min, then wash MeOH off with running buffer</div>
-</li>
-<li class="level1"><div class="li"> assembly: sponge, whatman paper, PVDF membrane, gel, whatman paper, sponge (every step flatten stack by rolling out from centre to outside)</div>
-</li>
-<li class="level1"><div class="li"> run 10-12 h const. 20mAmp (also possible: 1:35 h 44 Amp/45 V)</div>
-</li>
-</ul>
 </div>
-<div class='secedit editbutton_section editbutton_429'> </div>
+<div class='secedit editbutton_section editbutton_143'> </div>
-<h2 class="sectionedit430" id="section1707171">17.07.17</h2>
+<h2 class="sectionedit144" id="section151017">15.10.17</h2>
 <div class="level2">
 </div>
-<div class='secedit editbutton_section editbutton_430'> </div>
+<div class='secedit editbutton_section editbutton_144'> </div>
-<h3 class="sectionedit431" id="colonies_picked_from_cahif1a_cloning">Colonies picked from CaHIF1A cloning</h3>
+<h3 class="sectionedit145" id="pick_colonies_of_the_interlab_study">pick colonies of the Interlab Study</h3>
 <div class="level3">
-<p>
-colonies were picked.
-</p>
 </div>
-<div class='secedit editbutton_section editbutton_431'> </div>
+<div class='secedit editbutton_section editbutton_147'> </div>
-<h2 class="sectionedit432" id="section180817">18.08.17</h2>
+<h2 class="sectionedit148" id="section171017">17.10.17</h2>
 <div class="level2">
-</div>
+<p>
-<div class='secedit editbutton_section editbutton_432'> </div>
+<strong><em class="u">calibration plate reader</em></strong>
-<h3 class="sectionedit433" id="cahif1a_miniprep_test_digest_and_glycerol_stocks">CaHif1A miniprep, test digest and glycerol stocks</h3>
+</p>
-<div class="level3">
 <p>
-Miniprep was performed on overnight cultures CaHIF1A C1 and C2 according to manufacturer&#039;s protocol (zymo).<br/>
+-calibration of plate reader for CFP with dilutions of knockdown Jurkat and HEK cells (CMV_CFP)<br/>
-</p>
+-black 96 well plate with clear bottom<br/>
+-cells/well: 50k, 100k<br/>
-<p>
+-wavelengths: Ex.: 433±20 nm; Emm.: 475±9 nm
-C1 test digest (see 11.08.17)
 </p>
-<div class="table sectionedit434"><table class="inline">
+<div class="table sectionedit149"><table class="inline">
 	<thead>
 	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
+		<th class="col0">HEK</th><th class="col1">Jurkat</th>
 	</tr>
 	</thead>
 	<tr class="row1">
-		<td class="col0"> 1.2 µl </td><td class="col1"> plasmid (745 ng/µl)</td>
+		<td class="col0"><img style="max-height: 300px" src="https://static.igem.org/mediawiki/2017/2/26/T-Freiburg-10_17_2017_calibration_1.jpeg"<a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=cal_hek_kd.jpg" class="media" title="cal_hek_kd.jpg"><img src="/igem2017/lib/exe/fetch.php?w=400&amp;tok=1603e3&amp;media=cal_hek_kd.jpg" class="mediacenter" alt="" width="400" /></a></td><td class="col1"><img style="max-height: 300px" src="https://static.igem.org/mediawiki/2017/2/28/T-Freiburg-10_17_2017_calibration_2.jpeg"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=cal_jk_kd.jpg" class="media" title="cal_jk_kd.jpg"><img src="/igem2017/lib/exe/fetch.php?w=400&amp;tok=36dc3f&amp;media=cal_jk_kd.jpg" class="mediacenter" alt="" width="400" /></a></td>
-	</tr>
-	<tr class="row2">
-		<td class="col0"> 1 µl </td><td class="col1"> CutSmart </td>
-	</tr>
-	<tr class="row3">
-		<td class="col0"> 1 µl </td><td class="col1"> XhoI </td>
-	</tr>
-	<tr class="row4">
-		<td class="col0"> 6.8 µl </td><td class="col1"> H2O </td>
 	</tr>
 </table></div>
-<div class='secedit editbutton_table editbutton_434'> </div>
+<div class='secedit editbutton_table editbutton_149'> </div>
-<p>
+</div>
-C2 test digest
+<div class='secedit editbutton_section editbutton_148'> </div>
-</p>
+<h3 class="sectionedit150" id="cell_measurements_of_the_interlab_study">cell measurements of the Interlab Study</h3>
-<div class="table sectionedit435"><table class="inline">
+<div class="level3">
-	<thead>
-	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<td class="col0"> 3 µl </td><td class="col1"> plasmid (372 ng/µl)</td>
-	</tr>
-	<tr class="row2">
-		<td class="col0"> 1 µl </td><td class="col1"> CutSmart </td>
-	</tr>
-	<tr class="row3">
-		<td class="col0"> 1 µl </td><td class="col1"> XhoI </td>
-	</tr>
-	<tr class="row4">
-		<td class="col0"> 5 µl </td><td class="col1"> H2O </td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_435'> </div>
 <p>
-Glycerol stocks of CaHif1A were produced.<br/>
+- OD600 and fluorescence intensity of each device after 0h, 2h, 4h and 6h<br/>
-</p>
+- again random fluorescence values<br/>
-<p>
-<img src="https://static.igem.org/mediawiki/2017/e/ef/T%E2%80%93FREIBURG%E2%80%93658.png" width="500px">
 </p>
 </div>
-<div class='secedit editbutton_section editbutton_433'> </div>
+<div class='secedit editbutton_section editbutton_147'> </div>
-<h3 class="sectionedit436" id="section200817">20.08.17</h3>
+<h3 class="sectionedit148" id="pei_transfection_of_cmv-vegfr2_sv40-tdag8">PEI transfection of CMV-VEGFR2, SV40-TDAG8</h3>
 <div class="level3">
+<div class="table sectionedit149"><table class="inline">
-</div>
-<h4 id="ko-kit_confirm_pcr">KO-Kit confirm PCR</h4>
-<div class="level4">
-<p>
-To confirm knockout a PCR was performed.
-</p>
-<p>
-Mix 1
-</p>
-<div class="table sectionedit437"><table class="inline">
 	<thead>
 	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">volume</th><th class="col2">component</th>
+		<th class="col0">plasmid</th>
 	</tr>
 	</thead>
 	<tr class="row1">
-		<td class="col0"> 1 µl </td><td class="col1"> </td><td class="col2"> template </td>
+		<td class="col0">CMV-VEGFR2</td>
 	</tr>
 	<tr class="row2">
-		<td class="col0"> 1.5 µl </td><td class="col1"> 13.5 µl </td><td class="col2"> buffer 10x </td>
+		<td class="col0">SV40-TDAG8</td>
-	</tr>
-	<tr class="row3">
-		<td class="col0"> 1 µl </td><td class="col1"> 9 µl </td><td class="col2"> primer 228 </td>
-	</tr>
-	<tr class="row4">
-		<td class="col0"> 1 µl </td><td class="col1"> 9 µl </td><td class="col2"> primer 229 </td>
-	</tr>
-	<tr class="row5">
-		<td class="col0"> 1 µl </td><td class="col1"> 9 µl </td><td class="col2"> dNTPs </td>
-	</tr>
-	<tr class="row6">
-		<td class="col0"> 0.5 µl </td><td class="col1"> 4.5 µl </td><td class="col2"> Taq Q5 </td>
-	</tr>
-	<tr class="row7">
-		<td class="col0"> 9 µl </td><td class="col1"> 81 µl </td><td class="col2"> H2O </td>
 	</tr>
 </table></div>
-<div class='secedit editbutton_table editbutton_437'> </div>
+<div class='secedit editbutton_table editbutton_149'> </div><ol>
-<p>
+<li class="level1"><div class="li"> Split the cells 1:2 into 10 cm plate<br/>
-Mix 2
+</div>
-</p>
+</li>
-<div class="table sectionedit438"><table class="inline">
+<li class="level1"><div class="li"> Thaw PEI reagent at RT<br/>
-	<thead>
+</div>
-	<tr class="row0">
+</li>
-		<th class="col0">volume</th><th class="col1">volume</th><th class="col2">component</th>
+<li class="level1"><div class="li"> Dilute plasmid in 1 ml serum free DMEM (2.5 µg from each plasmid)<br/>
-	</tr>
+</div>
-	</thead>
+</li>
-	<tr class="row1">
+<li class="level1"><div class="li"> Add 15 µl PEI(1 µg/µl) to the mix while vortexing (PEI:DNA = 3:1)<br/>
-		<td class="col0"> 1 µl </td><td class="col1"> 9 µl </td><td class="col2"> template </td>
+</div>
-	</tr>
+</li>
-	<tr class="row2">
+<li class="level1"><div class="li"> incubate 15 min at RT<br/>
-		<td class="col0"> 1.5 µl </td><td class="col1"> 13.5 µl </td><td class="col2"> buffer 10x </td>
+</div>
-	</tr>
+</li>
-	<tr class="row3">
+<li class="level1"><div class="li"> Add the mix to the culture<br/>
-		<td class="col0"> 1 µl </td><td class="col1"> 9 µl </td><td class="col2"> primer 60 </td>
+</div>
-	</tr>
+</li>
-	<tr class="row4">
+<li class="level1"><div class="li"> incubate for 24 h<br/>
-		<td class="col0"> 1 µl </td><td class="col1"> 9 µl </td><td class="col2"> primer 32 </td>
+</div>
-	</tr>
+</li>
-	<tr class="row5">
+</ol>
-		<td class="col0"> 1 µl </td><td class="col1"> 9 µl </td><td class="col2"> dNTPs </td>
-	</tr>
-	<tr class="row6">
-		<td class="col0"> 0.5 µl </td><td class="col1"> 4.5 µl </td><td class="col2"> Taq Q5 </td>
-	</tr>
-	<tr class="row7">
-		<td class="col0"> 9 µl </td><td class="col1"> 81 µl </td><td class="col2"> H2O </td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_438'> </div>
-<p>
-Picture in lab book.<br/>
-</p>
+</div>
+<div class='secedit editbutton_section editbutton_148'> </div>
+<h2 class="sectionedit153" id="section181017">18.10.17</h2>
+<div class="level2">
 </div>
-<div class='secedit editbutton_section editbutton_436'> </div>
+<!-- EDIT153 SECTION "18.10.17" [29522-29542] -->
-<h3 class="sectionedit439" id="cell_lysis_of_jk_cells">Cell lysis of JK cells</h3>
+<h3 class="sectionedit154" id="input_tests_in_hek_and_jurkat_cells">input tests in HEK and Jurkat cells</h3>
 <div class="level3">
 <p>
-Lysis of JK cells was performed according to protocol.<br/>
+used medium: PBS (10% FCS, 0.5% glucose, 1% HEPES)<br/>
 </p>
-</div>
+<p>
-<div class='secedit editbutton_section editbutton_439'> </div>
+fluorescence measurement after 0h, 1h, 2h, 4h, 6h, 9h, 16, 24h<br/>
-<h2 class="sectionedit440" id="section210817">21.08.17</h2>
-<div class="level2">
-</div>
+</p>
-<div class='secedit editbutton_section editbutton_440'> </div>
-<h3 class="sectionedit441" id="sds-page_and_western_blot">SDS-PAGE and Western blot</h3>
-<div class="level3">
 <p>
-SDS-PAGE were ran for ~ 2 h, Western blots overnight (const. 20 mA, 4°C).
+in order to determine the relative fluorescence intensity (RFI) stable cell lines were substracted by wildtype cells with same treatment.<br/>
 </p>
-</div>
+<p>
-<div class='secedit editbutton_section editbutton_441'> </div>
+<strong>CRE(4x)-CFP-CMV-mCherry (stable), SV40-TDAG8 (transient):</strong><br/>
-<h2 class="sectionedit442" id="section220817">22.08.17</h2>
-<div class="level2">
-</div>
+</p>
-<div class='secedit editbutton_section editbutton_442'> </div>
-<h3 class="sectionedit443" id="blocking_western_blots">Blocking Western blots</h3>
-<div class="level3">
 <p>
-Blots were blocked for ~ 6 h, then stored at 4 °C (picture in lab book).<br/>
+conditions:
 </p>
+<div class="table sectionedit155"><table class="inline">
-</div>
-<div class='secedit editbutton_section editbutton_443'> </div>
-<h3 class="sectionedit444" id="generated_bsa_standard_in_tbs-t">Generated BSA standard in TBS-T</h3>
-<div class="level3">
-<div class="table sectionedit445"><table class="inline">
 	<thead>
 	<tr class="row0">
-		<th class="col0">BSA concentration</th><th class="col1">No.</th><th class="col2">lambda</th><th class="col3">Absorbance</th>
+		<th class="col0">pH</th><th class="col1">Forskolin + IBMX (100 µM each)</th><th class="col2">transient SV40_TDAG8 (HEK only)</th>
 	</tr>
 	</thead>
 	<tr class="row1">
-		<td class="col0"> 0 µg/ml </td><td class="col1"> 2 </td><td class="col2"> 620nm </td><td class="col3"> 0.005A </td>
+		<td class="col0">6.5</td><td class="col1">-</td><td class="col2">-</td>
 	</tr>
 	<tr class="row2">
-		<td class="col0"> 0 µg/ml </td><td class="col1"> 2 </td><td class="col2"> 620nm </td><td class="col3"> 0.007A </td>
+		<td class="col0">6.5</td><td class="col1">+</td><td class="col2">-</td>
 	</tr>
 	<tr class="row3">
-		<td class="col0"> 0 µg/ml </td><td class="col1"> 2 </td><td class="col2"> 620nm </td><td class="col3"> 0.007A </td>
+		<td class="col0">6.5</td><td class="col1">-</td><td class="col2">+</td>
 	</tr>
 	<tr class="row4">
-		<td class="col0"> 5 µg/ml </td><td class="col1"> 3 </td><td class="col2"> 620nm </td><td class="col3"> 0.140A </td>
+		<td class="col0">6.5</td><td class="col1">+</td><td class="col2">+</td>
 	</tr>
 	<tr class="row5">
-		<td class="col0"> 5 µg/ml </td><td class="col1"> 3 </td><td class="col2"> 620nm </td><td class="col3"> 0.161A </td>
+		<td class="col0">7.1</td><td class="col1">-</td><td class="col2">-</td>
 	</tr>
 	<tr class="row6">
-		<td class="col0"> 5 µg/ml </td><td class="col1"> 3 </td><td class="col2"> 620nm </td><td class="col3"> 0.115A </td>
+		<td class="col0">7.1</td><td class="col1">+</td><td class="col2">-</td>
 	</tr>
 	<tr class="row7">
-		<td class="col0"> 10 µg/ml </td><td class="col1"> 3 </td><td class="col2"> 620nm </td><td class="col3"> 0.275A </td>
+		<td class="col0">7.1</td><td class="col1">-</td><td class="col2">+</td>
 	</tr>
 	<tr class="row8">
-		<td class="col0"> 10 µg/ml </td><td class="col1"> 3 </td><td class="col2"> 620nm </td><td class="col3"> 0.284A </td>
+		<td class="col0">7.1</td><td class="col1">+</td><td class="col2">+</td>
 	</tr>
 	<tr class="row9">
-		<td class="col0"> 10 µg/ml </td><td class="col1"> 3 </td><td class="col2"> 620nm </td><td class="col3"> 0.271A </td>
+		<td class="col0">7.7</td><td class="col1">-</td><td class="col2">-</td>
 	</tr>
 	<tr class="row10">
-		<td class="col0"> 20 µg/ml </td><td class="col1"> 3 </td><td class="col2"> 620nm </td><td class="col3"> 0.581A </td>
+		<td class="col0">7.7</td><td class="col1">+</td><td class="col2">-</td>
 	</tr>
 	<tr class="row11">
-		<td class="col0"> 15 µg/ml </td><td class="col1"> 3 </td><td class="col2"> 620nm </td><td class="col3"> 0.435A </td>
+		<td class="col0">7.7</td><td class="col1">-</td><td class="col2">+</td>
 	</tr>
 	<tr class="row12">
-		<td class="col0"> 15 µg/ml </td><td class="col1"> 3 </td><td class="col2"> 620nm </td><td class="col3"> 0.429A </td>
+		<td class="col0">7.7</td><td class="col1">+</td><td class="col2">+</td>
 	</tr>
 	<tr class="row13">
-		<td class="col0"> 15 µg/ml </td><td class="col1"> 3 </td><td class="col2"> 620nm </td><td class="col3"> 0.430A </td>
+		<td class="col0">6.5 → 7.7</td><td class="col1">-</td><td class="col2">-</td>
 	</tr>
 	<tr class="row14">
-		<td class="col0"> 20 µg/ml </td><td class="col1"> 3 </td><td class="col2"> 620nm </td><td class="col3"> 0.545A </td>
+		<td class="col0">6.5 → 7.7</td><td class="col1">+</td><td class="col2">-</td>
 	</tr>
 	<tr class="row15">
-		<td class="col0"> 20 µg/ml </td><td class="col1"> 3 </td><td class="col2"> 620nm </td><td class="col3"> 0.574A </td>
+		<td class="col0">6.5 → 7.7</td><td class="col1">-</td><td class="col2">+</td>
 	</tr>
 	<tr class="row16">
-		<td class="col0"> 30 µg/ml </td><td class="col1"> 3 </td><td class="col2"> 620nm </td><td class="col3"> 0.830A </td>
+		<td class="col0">6.5 → 7.7</td><td class="col1">+</td><td class="col2">+</td>
-	</tr>
-	<tr class="row17">
-		<td class="col0"> 30 µg/ml </td><td class="col1"> 3 </td><td class="col2"> 620nm </td><td class="col3"> 0.886A </td>
-	</tr>
-	<tr class="row18">
-		<td class="col0"> 30 µg/ml </td><td class="col1"> 3 </td><td class="col2"> 620nm </td><td class="col3"> 0.887A </td>
 	</tr>
 </table></div>
-<div class='secedit editbutton_table editbutton_445'> </div>
+<!-- EDIT155 TABLE [29928-30184] -->
-</div>
-<div class='secedit editbutton_section editbutton_444'> </div>
-<h2 class="sectionedit446" id="section240817">24.08.17</h2>
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_446'> </div>
-<h3 class="sectionedit447" id="sds-page1">SDS-PAGE</h3>
-<div class="level3">
 <p>
-Protein concentration was determined using amido black stain. 10 µg protein for each sample were was loaded on gel (problem with CoCl2 treated sample: not able to load appropriate volume). Blotting occurred over night at 4 °C (20 mA, 12 h). Membrane was blocked with 5% milk powder in TBST for 2.5 h.<br/>
+<em class="u">Results:</em><br/>
 </p>
-<div class="table sectionedit448"><table class="inline">
+<div class="table sectionedit156"><table class="inline">
+	<thead>
 	<tr class="row0">
-		<td class="col0"> cell line </td><td class="col1"> JK </td><td class="col2"> JK </td><td class="col3"> JK </td><td class="col4"> JK </td><td class="col5"> HEK </td>
+		<th class="col0">HEK293T</th><th class="col1">Jurkat</th>
 	</tr>
+	</thead>
 	<tr class="row1">
-		<td class="col0"> CoCl2 (µM) </td><td class="col1"> 0 </td><td class="col2"> 200 </td><td class="col3"> 400 </td><td class="col4"> 800 </td><td class="col5"> 500 </td>
+		<td class="col0"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=hek_cre_tdag8_rfi.jpg" class="media" title="hek_cre_tdag8_rfi.jpg"><img src="https://static.igem.org/mediawiki/2017/a/a3/T%E2%80%93FREIBURG%E2%80%93Modeling-18.10.17-1.jpg" class="mediacenter" alt="" width="400" /></a></td><td class="col1"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=jk_cre_rfi.jpg" class="media" title="jk_cre_rfi.jpg"><img src="https://static.igem.org/mediawiki/2017/7/79/T%E2%80%93FREIBURG%E2%80%93Modeling-18.10.17-2.jpg" class="mediacenter" alt="" width="400" /></a></td>
 	</tr>
-</table></div>
+	<tr class="row2">
-<div class='secedit editbutton_table editbutton_448'> </div>
+		<td class="col0"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=hek_cre_6.5_rfi.jpg" class="media" title="hek_cre_6.5_rfi.jpg"><img src="https://static.igem.org/mediawiki/2017/f/f9/T%E2%80%93FREIBURG%E2%80%93Modeling-18.10.17-3.jpg" class="mediacenter" alt="" width="400" /></a></td><td class="col1"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=jk_cre_f_i_rfi.jpg" class="media" title="jk_cre_f_i_rfi.jpg"><img src="https://static.igem.org/mediawiki/2017/a/a8/T%E2%80%93FREIBURG%E2%80%93Modeling-18.10.17-4.jpg" class="mediacenter" alt="" width="400" /></a></td>
-</div>
-<div class='secedit editbutton_section editbutton_447'> </div>
-<h2 class="sectionedit449" id="section260817">26.08.17</h2>
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_449'> </div>
-<h3 class="sectionedit450" id="lenti-cas9-puro_all_rnas_electroporation">lenti-Cas9-puro all RNAs electroporation</h3>
-<div class="level3">
-<p>
-All RNA coding lenti-Cas9-puro were electroporated into Jurkat using CAS9-GFP RNAscramble as transfection control.<br/>
-Protocol from cell culture was used:
-</p>
-<ul>
-<li class="level1"><div class="li"> 2&times;10^6 cells and 9 µg DNA were resuspended in 200 µl 3P buffer</div>
-</li>
-<li class="level1"><div class="li"> put in cuvettes and electroporated</div>
-</li>
-<li class="level1"><div class="li"> 500 µl RPMI were added</div>
-</li>
-<li class="level1"><div class="li"> plated on 12 well plate, filled to 2 ml with RPMI</div>
-</li>
-</ul>
-</div>
-<div class='secedit editbutton_section editbutton_450'> </div>
-<h3 class="sectionedit451" id="puromycin_toxicity_assay">Puromycin toxicity assay</h3>
-<div class="level3">
-<p>
-HEK cells were transfected with lenti-Cas9-puro RNA5 old cloning and SGEN as transfection control using PEI (1x10cm dish at ~50% confluency). 1 ml serum, phenol red free DMEM and 10 µg DNA were vortexed. 30 µl PEI (1µg/ml) were added and vortexed carefully. After 15 min PEI/DNA was added evenly to dish.
-</p>
-</div>
-<div class='secedit editbutton_section editbutton_451'> </div>
-<h2 class="sectionedit452" id="section310817">31.08.17</h2>
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_452'> </div>
-<h3 class="sectionedit453" id="sds-page2">SDS-PAGE</h3>
-<div class="level3">
-<p>
-Protein amount was determined using amido black stain. 10 µg protein for each sample were loaded. SDS-Page took ~ 2 h and blotting 2 h at 250 mA. Membranes were blocked overnight at 4°C. No results could be observed.
-</p>
-<div class="table sectionedit454"><table class="inline">
-	<tr class="row0">
-		<td class="col0"> Cell line </td><td class="col1"> HEK </td><td class="col2"> HEK </td><td class="col3"> HEK </td><td class="col4"> JK </td><td class="col5"> JK </td><td class="col6"> JK </td><td class="col7"> JK </td>
 	</tr>
-	<tr class="row1">
+	<tr class="row3">
-		<td class="col0"> CoCl2 (µM) </td><td class="col1"> 0 </td><td class="col2"> 200 </td><td class="col3"> 500 </td><td class="col4"> 0 </td><td class="col5"> 400 </td><td class="col6"> 200 </td><td class="col7"> 800 </td>
+		<td class="col0"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=hek_cre_7.1_rfi.jpg" class="media" title="hek_cre_7.1_rfi.jpg"><img src="https://static.igem.org/mediawiki/2017/3/3a/T%E2%80%93FREIBURG%E2%80%93Modeling-18.10.17-5.jpg" class="mediacenter" alt="" width="400" /></a></td><td class="col1"> </td>
 	</tr>
-</table></div>
+	<tr class="row4">
-<div class='secedit editbutton_table editbutton_454'> </div>
+		<td class="col0"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=hek_cre_7.7_rfi.jpg" class="media" title="hek_cre_7.7_rfi.jpg"><img src="https://static.igem.org/mediawiki/2017/3/37/T%E2%80%93FREIBURG%E2%80%93Modeling-18.10.17-6.jpg" class="mediacenter" alt="" width="400" /></a></td><td class="col1"> </td>
-</div>
-<div class='secedit editbutton_section editbutton_453'> </div>
-              </div>
-           <button class="accordion">September</button>
-        <div id="vanish" class="panel panel-default">
-<h2 class="sectionedit455" id="section030917">03.09.17</h2>
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_455'> </div>
-<h3 class="sectionedit456" id="sds-page_with_new_samples">SDS-PAGE with new samples</h3>
-<div class="level3">
-<p>
-Treating blots made on 31.08.17 with new ECL showed no results. Western blots were repeated but without success.
-</p>
-</div>
-<div class='secedit editbutton_section editbutton_456'> </div>
-<h2 class="sectionedit457" id="section100917">10.09.17</h2>
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_457'> </div>
-<h3 class="sectionedit458" id="cell_lysis_and_determining_protein_concentration">Cell lysis and determining protein concentration</h3>
-<div class="level3">
-<p>
-Cell lysis was performed according to protocol (6cm dish JK with 150 µl RIPA, 6 well HEK in 250 µl RIPA). Protein concentration was determined using amido black stain.
-</p>
-</div>
-<div class='secedit editbutton_section editbutton_458'> </div>
-<h2 class="sectionedit459" id="section110917">11.09.17</h2>
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_459'> </div>
-<h3 class="sectionedit460" id="sds-page_and_western_blot1">SDS-PAGE and Western Blot</h3>
-<div class="level3">
-<p>
-µg protein for each sample were loaded on gel for SDS-PAGE (0.02 A in stacking-gel, 0.05 A in running-gel). Blotting was performed at 250mA for 2 h. Blocking occured at room temperature for 2 h in 5% milk-TBS-T. Blots were incubated with primary antibodies overnight (anti-HIF1A, anti-GAPDH). <br/>
-</p>
-<div class="table sectionedit461"><table class="inline">
-	<tr class="row0">
-		<td class="col0"> cell line </td><td class="col1"> JK </td><td class="col2"> JK </td><td class="col3"> JK </td><td class="col4"> JK </td><td class="col5"> HEK </td><td class="col6"> HEK </td><td class="col7"> HEK </td><td class="col8"> HEK </td><td class="col9"> HEK </td>
 	</tr>
-	<tr class="row1">
+	<tr class="row5">
-		<td class="col0"> CoCl2 (µM) </td><td class="col1"> 0 </td><td class="col2"> 200 </td><td class="col3"> 400 </td><td class="col4"> 800 </td><td class="col5"> 0 </td><td class="col6"> 200 </td><td class="col7"> 400 </td><td class="col8"> 800 </td><td class="col9"> 800 </td>
+		<td class="col0"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=hek_cre_7.7_adj_rfi.jpg" class="media" title="hek_cre_7.7_adj_rfi.jpg"><img src="https://static.igem.org/mediawiki/2017/8/8f/T%E2%80%93FREIBURG%E2%80%93Modeling-18.10.17-7.jpg" class="mediacenter" alt="" width="400" /></a></td><td class="col1"> </td>
 	</tr>
 </table></div>
-<div class='secedit editbutton_table editbutton_461'> </div>
+<!-- EDIT156 TABLE [30201-30464] -->
-</div>
-<div class='secedit editbutton_section editbutton_460'> </div>
-<h2 class="sectionedit462" id="section120917">12.09.17</h2>
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_462'> </div>
-<h3 class="sectionedit463" id="washing_blots">Washing blots</h3>
-<div class="level3">
 <p>
-Blots were washed with TBS-T 3x10min and incubated with 1:10,000 secondary antibody for 1 h (anti-mouse HRp –&gt; HIF1A; anti-rabbit HRP –&gt; GAPDH).
+<strong>Ctla4(4x)-CFP-CMV-mCherry (stable), CMV-VEGFR-2 (transient):
-</p>
+</strong><br/>
-</div>
-<div class='secedit editbutton_section editbutton_463'> </div>
-<h2 class="sectionedit464" id="section140917">14.09.17</h2>
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_464'> </div>
-<h3 class="sectionedit465" id="lysis_of_jk_cells_and_determining_protein_concentration">Lysis of JK cells and determining protein concentration</h3>
-<div class="level3">
-<p>
-JK cells were induced with CoCl2 24 h before lysis. Lysis was performed in 150 µl RIPA and 100 µM CoCl2. Protein concentration was determined with amido black stain.
 </p>
-</div>
-<div class='secedit editbutton_section editbutton_465'> </div>
-<h2 class="sectionedit466" id="section150917">15.09.17</h2>
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_466'> </div>
-<h3 class="sectionedit467" id="sds-gel_for_jk">SDS-Gel for JK</h3>
-<div class="level3">
 <p>
-Samples were collected one day prior to running the gel. Lysis was performed according to protocol. Amido black stain was used to determine protein amount. Proteins were blotted for 2 h at 250 mA. The mambrane was blocked for 2 h at room temperature.
+conditions:
 </p>
-<div class="table sectionedit468"><table class="inline">
+<div class="table sectionedit157"><table class="inline">
+	<thead>
 	<tr class="row0">
-		<td class="col0">cell line </td><td class="col1"> ladder </td><td class="col2"> JK </td><td class="col3"> JK </td><td class="col4"> JK </td><td class="col5"> JK </td><td class="col6"> JK </td><td class="col7"> JK </td>
+		<th class="col0">VEGF (ng/ml)</th><th class="col1">Ionomycin (µmol/l)</th><th class="col2">transient CMV-VEGFR-2 (HEK only)</th>
 	</tr>
+	</thead>
 	<tr class="row1">
-		<td class="col0"> CoCl2 (µM) </td><td class="col1 leftalign">  </td><td class="col2"> 0 </td><td class="col3"> 20 </td><td class="col4"> 40 </td><td class="col5"> 80 </td><td class="col6"> 160 </td><td class="col7"> 200 </td>
+		<td class="col0">0</td><td class="col1"> </td><td class="col2">+</td>
 	</tr>
-</table></div>
+	<tr class="row2">
-<div class='secedit editbutton_table editbutton_468'> </div>
+		<td class="col0">0</td><td class="col1"> </td><td class="col2">-</td>
-</div>
-<div class='secedit editbutton_section editbutton_467'> </div>
-<h2 class="sectionedit469" id="section170917">17.09.17</h2>
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_469'> </div>
-<h3 class="sectionedit470" id="sds-page3">SDS-PAGE</h3>
-<div class="level3">
-<p>
-Gel performed according to protocol. Western blot was performed for 2 h at 250 mA. Protein amount was determined using amido black stain. Blots were blocked for 2 h at room temperature and primary antibody was added over night.<br/>
-</p>
-<div class="table sectionedit471"><table class="inline">
-	<tr class="row0">
-		<td class="col0">cell line </td><td class="col1"> ladder </td><td class="col2"> HEK:133 </td><td class="col3"> HEK:133 </td><td class="col4"> HEK:133 </td><td class="col5"> HEK:133 </td><td class="col6"> HEK:WT </td><td class="col7"> HEK:WT </td><td class="col8"> HEK:WT </td>
 	</tr>
-	<tr class="row1">
+	<tr class="row3">
-		<td class="col0"> CoCl2 (µM) </td><td class="col1 leftalign">  </td><td class="col2"> 0 </td><td class="col3"> 200 </td><td class="col4"> 400 </td><td class="col5"> 600 </td><td class="col6"> 0 </td><td class="col7"> 400 </td><td class="col8"> 600 </td>
+		<td class="col0">25</td><td class="col1"> </td><td class="col2">+</td>
 	</tr>
-</table></div>
+	<tr class="row4">
-<div class='secedit editbutton_table editbutton_471'> </div><div class="table sectionedit472"><table class="inline">
+		<td class="col0">25</td><td class="col1"> </td><td class="col2">-</td>
-	<tr class="row0">
-		<td class="col0">cell line </td><td class="col1"> ladder </td><td class="col2"> HEK:134 </td><td class="col3"> HEK:134 </td><td class="col4"> HEK:134 </td><td class="col5"> HEK:134 </td><td class="col6"> HEK:WT </td><td class="col7"> HEK:WT </td><td class="col8"> HEK:WT </td>
 	</tr>
-	<tr class="row1">
+	<tr class="row5">
-		<td class="col0"> CoCl2 (µM) </td><td class="col1 leftalign">  </td><td class="col2"> 0 </td><td class="col3"> 200 </td><td class="col4"> 400 </td><td class="col5"> 600 </td><td class="col6"> 0 </td><td class="col7"> 400 </td><td class="col8"> 600 </td>
+		<td class="col0">50</td><td class="col1"> </td><td class="col2">+</td>
 	</tr>
-</table></div>
+	<tr class="row6">
-<div class='secedit editbutton_table editbutton_472'> </div>
+		<td class="col0">50</td><td class="col1"> </td><td class="col2">-</td>
-</div>
-<div class='secedit editbutton_section editbutton_470'> </div>
-<h2 class="sectionedit473" id="section180917">18.09.17</h2>
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_473'> </div>
-<h3 class="sectionedit474" id="sds-page_and_western_blot2">SDS-PAGE and Western Blot</h3>
-<div class="level3">
-<p>
-SDS-PAGE and Western Blot were performed according to protocol<br/>
-</p>
-<div class="table sectionedit475"><table class="inline">
-	<tr class="row0">
-		<td class="col0"> cell line </td><td class="col1"> ladder </td><td class="col2"> JK:133 </td><td class="col3"> JK:133 </td><td class="col4"> JK:133 </td><td class="col5"> JK:133 </td><td class="col6"> JK:WT </td><td class="col7"> JK:WT </td><td class="col8"> JK:WT </td><td class="col9"> JK:WT </td><td class="col10"> HEK </td>
 	</tr>
-	<tr class="row1">
+	<tr class="row7">
-		<td class="col0"> CoCl2 (µM) </td><td class="col1 leftalign">  </td><td class="col2"> 0 </td><td class="col3"> 80 </td><td class="col4"> 160 </td><td class="col5"> 200 </td><td class="col6"> 0 </td><td class="col7"> 80 </td><td class="col8"> 160 </td><td class="col9"> 200 </td><td class="col10"> 600 </td>
+		<td class="col0">100</td><td class="col1"> </td><td class="col2">+</td>
 	</tr>
-</table></div>
+	<tr class="row8">
-<div class='secedit editbutton_table editbutton_475'> </div><div class="table sectionedit476"><table class="inline">
+		<td class="col0">100</td><td class="col1"> </td><td class="col2">-</td>
-	<tr class="row0">
-		<td class="col0"> cell line </td><td class="col1"> ladder </td><td class="col2"> HEK </td><td class="col3"> JK:133 </td><td class="col4"> JK:133 </td><td class="col5"> JK:133 </td><td class="col6"> JK:133 </td><td class="col7"> JK:WT </td><td class="col8"> JK:WT </td><td class="col9"> JK:WT </td><td class="col10"> JK:WT </td>
 	</tr>
-	<tr class="row1">
+	<tr class="row9">
-		<td class="col0"> CoCl2 (µM) </td><td class="col1 leftalign">  </td><td class="col2"> 600 </td><td class="col3"> 0 </td><td class="col4"> 80 </td><td class="col5"> 160 </td><td class="col6"> 200 </td><td class="col7"> 0 </td><td class="col8"> 80 </td><td class="col9"> 160 </td><td class="col10"> 200 </td>
+		<td class="col0"> </td><td class="col1">1.25</td><td class="col2">+</td>
 	</tr>
-</table></div>
+	<tr class="row10">
-<div class='secedit editbutton_table editbutton_476'> </div>
+		<td class="col0"> </td><td class="col1">1.25</td><td class="col2">-</td>
-</div>
-<div class='secedit editbutton_section editbutton_474'> </div>
-<h2 class="sectionedit477" id="section200917">20.09.17</h2>
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_477'> </div>
-<h3 class="sectionedit478" id="electroporation_of_5_grnas_into_jurkat">Electroporation of 5 gRNAs into Jurkat</h3>
-<div class="level3">
-<p>
-Electroporation of 5 gRNAs into Jurkat cells was performed according to protocol. GFP marker was used for sorting.
-</p>
-</div>
-<div class='secedit editbutton_section editbutton_478'> </div>
-<h2 class="sectionedit479" id="section210917">21.09.17</h2>
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_479'> </div>
-<h3 class="sectionedit480" id="electroporation_of_5_grnas_into_jurkat1">Electroporation of 5 gRNAs into Jurkat</h3>
-<div class="level3">
-<p>
-Electroporation of 5 gRNAs into Jurkat cells was repeated. GFP marker was used for sorting.
-</p>
-</div>
-<div class='secedit editbutton_section editbutton_480'> </div>
-<h3 class="sectionedit481" id="sds-page_and_western_blot3">SDS-PAGE and Western Blot</h3>
-<div class="level3">
-<p>
-SDS-PAGE and Western Blot were performed according to protocol.<br/>
-</p>
-<div class="table sectionedit482"><table class="inline">
-	<tr class="row0">
-		<td class="col0"> cell line </td><td class="col1"> ladder </td><td class="col2">HEK:WT </td><td class="col3"> HEK:WT </td><td class="col4"> HEK:WT </td><td class="col5"> HEK:133 </td><td class="col6"> HEK:133 </td><td class="col7"> HEK:133 </td><td class="col8"> HEK:133 </td>
 	</tr>
-	<tr class="row1">
+	<tr class="row11">
-		<td class="col0"> CoCl2 (µM) </td><td class="col1 leftalign">  </td><td class="col2"> 0 </td><td class="col3"> 400 </td><td class="col4"> 600 </td><td class="col5"> 0 </td><td class="col6"> 200 </td><td class="col7"> 400 </td><td class="col8"> 600 </td>
+		<td class="col0"> </td><td class="col1">2.5</td><td class="col2">+</td>
 	</tr>
-</table></div>
+	<tr class="row12">
-<div class='secedit editbutton_table editbutton_482'> </div><div class="table sectionedit483"><table class="inline">
+		<td class="col0"> </td><td class="col1">2.5</td><td class="col2">-</td>
-	<tr class="row0">
-		<td class="col0"> cell line </td><td class="col1"> ladder </td><td class="col2">HEK:WT </td><td class="col3"> HEK:WT </td><td class="col4"> HEK:WT </td><td class="col5"> HEK:134 </td><td class="col6"> HEK:134 </td><td class="col7"> HEK:134 </td><td class="col8"> HEK:134 </td>
 	</tr>
-	<tr class="row1">
+	<tr class="row13">
-		<td class="col0"> CoCl2 (µM) </td><td class="col1 leftalign">  </td><td class="col2"> 0 </td><td class="col3"> 400 </td><td class="col4"> 600 </td><td class="col5"> 0 </td><td class="col6"> 200 </td><td class="col7"> 400 </td><td class="col8"> 600 </td>
+		<td class="col0"> </td><td class="col1">5.0</td><td class="col2">+</td>
 	</tr>
-</table></div>
+	<tr class="row14">
-<div class='secedit editbutton_table editbutton_483'> </div>
+		<td class="col0"> </td><td class="col1">5.0</td><td class="col2">-</td>
-</div>
-<div class='secedit editbutton_section editbutton_481'> </div>
-<h2 class="sectionedit484" id="section220917">22.09.17</h2>
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_484'> </div>
-<h3 class="sectionedit485" id="sorting_gfp-positive_cells_of_pooled_electroporations">Sorting GFP-positive cells of pooled electroporations</h3>
-<div class="level3">
-<p>
-GFP-positive cells of pooled electroporations were sorted. Negative control was not mock electroporated and cound not be used for gating, hence data were gated based on samples. Single cells were seeded in 96-well plates into 50 µl mycoplasm clearance medium. Microscopy was used to check for presence of cells.
-</p>
-</div>
-<div class='secedit editbutton_section editbutton_485'> </div>
-<h2 class="sectionedit486" id="section230917">23.09.17</h2>
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_486'> </div>
-<h3 class="sectionedit487" id="sds-page_and_western_blot4">SDS-PAGE and Western Blot</h3>
-<div class="level3">
-<p>
-Samples were collected one day prior to SDS-PAGE gel. Protein amount was determined by amido black stain. SDS-PAGE and Western Blot were performed according to protocol (2 gels).<br/>
-</p>
-<div class="table sectionedit488"><table class="inline">
-	<tr class="row0">
-		<td class="col0"> cell line </td><td class="col1"> ladder </td><td class="col2"> JK:WT </td><td class="col3"> JK:WT </td><td class="col4"> JK:WT </td><td class="col5"> JK:WT </td><td class="col6"> JK:133 </td><td class="col7"> JK:133 </td><td class="col8"> JK:133 </td><td class="col9"> JK:133 </td>
 	</tr>
-	<tr class="row1">
+	<tr class="row15">
-		<td class="col0"> CoCl2 (µM) </td><td class="col1"> </td><td class="col2"> 0 </td><td class="col3"> 40 </td><td class="col4"> 80 </td><td class="col5"> 200 </td><td class="col6"> 0 </td><td class="col7"> 40 </td><td class="col8"> 80 </td><td class="col9"> 200 </td>
+		<td class="col0"> </td><td class="col1">10.0</td><td class="col2">+</td>
 	</tr>
-</table></div>
+	<tr class="row16">
-<div class='secedit editbutton_table editbutton_488'> </div>
+		<td class="col0"> </td><td class="col1">10.0</td><td class="col2">-</td>
-</div>
-<div class='secedit editbutton_section editbutton_487'> </div>
-<h2 class="sectionedit489" id="section240917">24.09.17</h2>
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_489'> </div>
-<h3 class="sectionedit490" id="adding_medium_to_seeded_single_cell_plates">Adding medium to seeded single cell plates</h3>
-<div class="level3">
-<p>
-µl conditioned RPMI was added to seeded single cell plates. In the following weeks regular check of clonal cultures and addition of conditioned RPMI if necessary.
-</p>
-</div>
-<div class='secedit editbutton_section editbutton_490'> </div>
-<h3 class="sectionedit491" id="sds-page_and_western_blot5">SDS-PAGE and Western Blot</h3>
-<div class="level3">
-<p>
-Samples were collected on 23.09.17. Protein amount was determined by amido black stain. Lysis of cells, SDS-PAGE and Western Blot were performed according to protocol. <br/>
-</p>
-<div class="table sectionedit492"><table class="inline">
-	<tr class="row0">
-		<td class="col0">cell line </td><td class="col1"> ladder </td><td class="col2"> HEK:WT </td><td class="col3"> HEK:WT</td><td class="col4"> HEK:WT </td><td class="col5"> HEK:133 </td><td class="col6"> HEK:133 </td><td class="col7"> HEK:133 </td><td class="col8"> HEK:133 </td>
-	</tr>
-	<tr class="row1">
-		<td class="col0"> CoCl2 (µM) </td><td class="col1 leftalign">  </td><td class="col2"> 0</td><td class="col3"> 400 </td><td class="col4"> 600 </td><td class="col5"> 0 </td><td class="col6">200 </td><td class="col7"> 400 </td><td class="col8">600 </td>
 	</tr>
 </table></div>
-<div class='secedit editbutton_table editbutton_492'> </div><div class="table sectionedit493"><table class="inline">
+<!-- EDIT157 TABLE [30548-30771] -->
-	<tr class="row0">
-		<td class="col0">cell line</td><td class="col1"> ladder </td><td class="col2"> HEK:WT </td><td class="col3"> HEK:WT</td><td class="col4"> HEK:WT </td><td class="col5"> HEK:134 </td><td class="col6"> HEK:134 </td><td class="col7"> HEK:134 </td><td class="col8"> HEK:134 </td>
-	</tr>
-	<tr class="row1">
-		<td class="col0"> CoCl2 (µM) </td><td class="col1 leftalign">  </td><td class="col2"> 0</td><td class="col3"> 400 </td><td class="col4"> 600 </td><td class="col5"> 0 </td><td class="col6">200 </td><td class="col7"> 400 </td><td class="col8">600 </td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_493'> </div>
-</div>
-<div class='secedit editbutton_section editbutton_491'> </div>
-<h2 class="sectionedit494" id="section290917">29.09.17</h2>
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_494'> </div>
-<h3 class="sectionedit495" id="sds-page_and_western_blot6">SDS-PAGE and Western Blot</h3>
-<div class="level3">
 <p>
-Experiments were performed according to protocol (see day before). CMV:HIF served as HA positive control.<br/>
+<em class="u">Results:</em>
 </p>
+<div class="table sectionedit158"><table class="inline">
-<p>
+	<thead>
-anti-HA
-</p>
-<div class="table sectionedit496"><table class="inline">
 	<tr class="row0">
-		<td class="col0"> cell line </td><td class="col1"> WT(HEK) </td><td class="col2"> WT(HEK) </td><td class="col3"> WT(HEK) </td><td class="col4"> WT(HEK) </td><td class="col5"> 133(HEK) </td><td class="col6"> 133(HEK) </td><td class="col7"> 133(HEK) </td><td class="col8"> 133(HEK) </td>
+		<th class="col0">HEK293T</th><th class="col1">Jurkat</th>
 	</tr>
+	</thead>
 	<tr class="row1">
-		<td class="col0"> CMV:HIF </td><td class="col1"> - </td><td class="col2"> - </td><td class="col3"> + </td><td class="col4"> + </td><td class="col5"> - </td><td class="col6"> - </td><td class="col7"> + </td><td class="col8"> + </td>
+		<td class="col0"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=hek_ctla4_ionomycin_rfi.jpg" class="media" title="hek_ctla4_ionomycin_rfi.jpg"><img src="https://static.igem.org/mediawiki/2017/a/af/T%E2%80%93FREIBURG%E2%80%93Modeling-18.10.17-8.jpg" class="mediacenter" alt="" width="400" /></a></td><td class="col1"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=jk_ctla4_ionomycin_rfi.jpg" class="media" title="jk_ctla4_ionomycin_rfi.jpg"><img src="https://static.igem.org/mediawiki/2017/c/c3/T%E2%80%93FREIBURG%E2%80%93Modeling-18.10.17-12.jpg" class="mediacenter" alt="" width="400" /></a></td>
 	</tr>
 	<tr class="row2">
-		<td class="col0"> CoCl2 (µM) </td><td class="col1"> 0 </td><td class="col2"> 400 </td><td class="col3"> 0 </td><td class="col4"> 400 </td><td class="col5"> 0 </td><td class="col6"> 400 </td><td class="col7"> 0 </td><td class="col8"> 400 </td>
+		<td class="col0"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=hek_ctla4_vegf_rfi.jpg" class="media" title="hek_ctla4_vegf_rfi.jpg"><img src="https://static.igem.org/mediawiki/2017/5/51/T%E2%80%93FREIBURG%E2%80%93Modeling-18.10.17-13.jpg" class="mediacenter" alt="" width="400" /></a></td><td class="col1"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=jk_ctla4_vegf_rfi.jpg" class="media" title="jk_ctla4_vegf_rfi.jpg"><img src="https://static.igem.org/mediawiki/2017/2/2f/T%E2%80%93FREIBURG%E2%80%93Modeling-18.10.17-14.jpg" class="mediacenter" alt="" width="400" /></a></td>
 	</tr>
-</table></div>
+	<tr class="row3">
-<div class='secedit editbutton_table editbutton_496'> </div><div class="table sectionedit497"><table class="inline">
+		<td class="col0"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=hek_ctla4_vegfr2_ionomycin_rfi.jpg" class="media" title="hek_ctla4_vegfr2_ionomycin_rfi.jpg"><img src="https://static.igem.org/mediawiki/2017/1/12/T%E2%80%93FREIBURG%E2%80%93Modeling-18.10.17-15.jpg" class="mediacenter" alt="" width="400" /></a></td><td class="col1"> </td>
-	<tr class="row0">
-		<td class="col0"> cell line </td><td class="col1"> WT(HEK) </td><td class="col2"> WT(HEK) </td><td class="col3"> WT(HEK) </td><td class="col4"> WT(HEK) </td><td class="col5"> 134(HEK) </td><td class="col6"> 134(HEK) </td><td class="col7"> 134(HEK) </td><td class="col8"> 134(HEK) </td>
 	</tr>
-	<tr class="row1">
+	<tr class="row4">
-		<td class="col0"> CMV:HIF </td><td class="col1"> - </td><td class="col2"> - </td><td class="col3"> + </td><td class="col4"> + </td><td class="col5"> - </td><td class="col6"> - </td><td class="col7"> + </td><td class="col8"> + </td>
+		<td class="col0"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=hek_ctla4_vegfr2_vegf_rfi.jpg" class="media" title="hek_ctla4_vegfr2_vegf_rfi.jpg"><img src="https://static.igem.org/mediawiki/2017/5/5e/T%E2%80%93FREIBURG%E2%80%93Modeling-18.10.17-16.jpg" class="mediacenter" alt="" width="400" /></a></td><td class="col1"> </td>
-	</tr>
-	<tr class="row2">
-		<td class="col0"> CoCl2 (µM) </td><td class="col1"> 0 </td><td class="col2"> 400 </td><td class="col3"> 0 </td><td class="col4"> 400 </td><td class="col5"> 0 </td><td class="col6"> 400 </td><td class="col7"> 0 </td><td class="col8"> 400 </td>
 	</tr>
 </table></div>
-<div class='secedit editbutton_table editbutton_497'> </div>
+<!-- EDIT158 TABLE [30787-31058] -->
-<p>
-anti-HIF1A
-</p>
-<div class="table sectionedit498"><table class="inline">
-	<tr class="row0">
-		<td class="col0"> cell line </td><td class="col1"> WT(HEK) </td><td class="col2"> WT(HEK) </td><td class="col3"> WT(HEK) </td><td class="col4"> WT(HEK) </td><td class="col5"> 133(HEK) </td><td class="col6"> 133(HEK) </td><td class="col7"> 133(HEK) </td><td class="col8"> 133(HEK) </td>
-	</tr>
-	<tr class="row1">
-		<td class="col0"> CMV:HIF </td><td class="col1"> - </td><td class="col2"> - </td><td class="col3"> + </td><td class="col4"> + </td><td class="col5"> - </td><td class="col6"> - </td><td class="col7"> + </td><td class="col8"> + </td>
-	</tr>
-	<tr class="row2">
-		<td class="col0"> CoCl2 (µM) </td><td class="col1"> 0 </td><td class="col2"> 400 </td><td class="col3"> 0 </td><td class="col4"> 400 </td><td class="col5"> 0 </td><td class="col6"> 400 </td><td class="col7"> 0 </td><td class="col8"> 400 </td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_498'> </div><div class="table sectionedit499"><table class="inline">
-	<tr class="row0">
-		<td class="col0"> cell line </td><td class="col1"> WT(HEK) </td><td class="col2"> WT(HEK) </td><td class="col3"> WT(HEK) </td><td class="col4"> WT(HEK) </td><td class="col5"> 134(HEK) </td><td class="col6"> 134(HEK) </td><td class="col7"> 134(HEK) </td><td class="col8"> 134(HEK) </td>
-	</tr>
-	<tr class="row1">
-		<td class="col0"> CMV:HIF </td><td class="col1"> - </td><td class="col2"> - </td><td class="col3"> + </td><td class="col4"> + </td><td class="col5"> - </td><td class="col6"> - </td><td class="col7"> + </td><td class="col8"> + </td>
-	</tr>
-	<tr class="row2">
-		<td class="col0"> CoCl2 (µM) </td><td class="col1"> 0 </td><td class="col2"> 400 </td><td class="col3"> 0 </td><td class="col4"> 400 </td><td class="col5"> 0 </td><td class="col6"> 400 </td><td class="col7"> 0 </td><td class="col8"> 400 </td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_499'> </div>
-</div>
-<div class='secedit editbutton_section editbutton_495'> </div>
-<h2 class="sectionedit500" id="section300917">30.09.17</h2>
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_500'> </div>
-<h3 class="sectionedit501" id="sample_preparation_sds-page_and_western_blot">Sample preparation, SDS-PAGE and Western Blot</h3>
-<div class="level3">
 <p>
-Sample preparation, SDS-PAGE and Western Blot as described earlier.<br/>
+<strong>HRE(4x)-CFP-CMV-mCherry (stable):
+</strong><br/>
 </p>
-<div class="table sectionedit502"><table class="inline">
-	<tr class="row0">
-		<td class="col0"> cell line treatment </td><td class="col1"> WT </td><td class="col2"> WT </td><td class="col3"> 134 </td><td class="col4"> 134 </td><td class="col5">134 </td><td class="col6"> 134 </td><td class="col7"> ladder </td><td class="col8"> 134 + CRE + Forskolin </td><td class="col9"> 134 + CRE + TDAG, pH7.5 </td><td class="col10"> 134 + CRE + TDAG, pH7.5 </td>
-	</tr>
-	<tr class="row1">
-		<td class="col0"> CMV:HIF </td><td class="col1"> - </td><td class="col2"> - </td><td class="col3"> - </td><td class="col4"> - </td><td class="col5"> + </td><td class="col6"> + </td><td class="col7 leftalign">  </td><td class="col8"> - </td><td class="col9"> - </td><td class="col10"> - </td>
-	</tr>
-	<tr class="row2">
-		<td class="col0"> CoCl2 (µM) </td><td class="col1"> 0 </td><td class="col2"> 400 </td><td class="col3"> 0 </td><td class="col4"> 400 </td><td class="col5">0 </td><td class="col6"> 400 </td><td class="col7 leftalign">  </td><td class="col8"> 0 </td><td class="col9"> 0 </td><td class="col10"> 400 </td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_502'> </div><div class="table sectionedit503"><table class="inline">
-	<tr class="row0">
-		<td class="col0"> cell line treatment </td><td class="col1"> WT </td><td class="col2"> WT </td><td class="col3"> 134 </td><td class="col4"> 134 </td><td class="col5">134 </td><td class="col6"> 134 </td><td class="col7"> ladder </td><td class="col8"> 134 + CRE + Forskolin </td><td class="col9"> 134 + CRE + TDAG, pH6.5 </td><td class="col10">134 + CRE + TDAG, pH6.5 </td>
-	</tr>
-	<tr class="row1">
-		<td class="col0"> CMV:HIF </td><td class="col1"> - </td><td class="col2"> - </td><td class="col3"> - </td><td class="col4"> - </td><td class="col5"> + </td><td class="col6"> + </td><td class="col7 leftalign">  </td><td class="col8"> - </td><td class="col9"> - </td><td class="col10"> - </td>
-	</tr>
-	<tr class="row2">
-		<td class="col0"> CoCl2 (µM) </td><td class="col1"> 0 </td><td class="col2"> 400 </td><td class="col3"> 0 </td><td class="col4"> 400 </td><td class="col5">0 </td><td class="col6"> 400 </td><td class="col7 leftalign">  </td><td class="col8"> 400 </td><td class="col9"> 0 </td><td class="col10"> 400 </td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_503'> </div>
-</div>
-<div class='secedit editbutton_section editbutton_501'> </div>
-              </div>
-              <button class="accordion">October</button>
-        <div id="vanish" class="panel panel-default">
-<h2 class="sectionedit504" id="section041017">04.10.17</h2>
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_504'> </div>
-<h3 class="sectionedit505" id="sample_preparation_sds-page_and_western_blot1">Sample preparation, SDS-PAGE and Western Blot</h3>
-<div class="level3">
 <p>
-Experiments were performed as described earlier.
+conditions:
 </p>
-<div class="table sectionedit506"><table class="inline">
+<div class="table sectionedit159"><table class="inline">
-	<tr class="row0">
-		<td class="col0"> cell line </td><td class="col1"> ladder </td><td class="col2"> WT </td><td class="col3"> WT </td><td class="col4"> WT </td><td class="col5"> WT </td><td class="col6"> 130 </td><td class="col7"> 130 </td><td class="col8"> 130 </td><td class="col9"> 130 </td>
-	</tr>
-	<tr class="row1">
-		<td class="col0"> CoCl2 (µM) </td><td class="col1 leftalign">  </td><td class="col2"> 0 </td><td class="col3"> 200 </td><td class="col4"> 400 </td><td class="col5"> 600 </td><td class="col6"> 0 </td><td class="col7"> 200 </td><td class="col8"> 400 </td><td class="col9"> 600 </td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_506'> </div>
-</div>
-<div class='secedit editbutton_section editbutton_505'> </div>
-<h2 class="sectionedit507" id="section051017">05.10.17</h2>
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_507'> </div>
-<h3 class="sectionedit508" id="sample_preparation_sds-page_and_western_blot2">Sample preparation, SDS-PAGE and Western Blot</h3>
-<div class="level3">
-<p>
-Experiments were performed as described earlier.
-</p>
-<div class="table sectionedit509"><table class="inline">
-	<tr class="row0">
-		<td class="col0"> cell line </td><td class="col1"> WT </td><td class="col2"> WT </td><td class="col3 rightalign">  WT </td><td class="col4"> 130 </td><td class="col5"> 130 </td><td class="col6"> 130 </td><td class="col7"> ladder </td><td class="col8"> GFP positive </td>
-	</tr>
-	<tr class="row1">
-		<td class="col0"> CoCl2 (µM) </td><td class="col1"> 0 </td><td class="col2"> 100 </td><td class="col3"> 200 </td><td class="col4"> 0 </td><td class="col5"> 100 </td><td class="col6"> 200 </td><td class="col7"> </td><td class="col8"> </td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_509'> </div><div class="table sectionedit510"><table class="inline">
-	<tr class="row0">
-		<td class="col0"> cell line </td><td class="col1"> ladder </td><td class="col2"> WT </td><td class="col3"> WT </td><td class="col4"> WT </td><td class="col5"> WT </td><td class="col6"> 130 </td><td class="col7"> 130 </td><td class="col8"> 130 </td><td class="col9"> 130 </td>
-	</tr>
-	<tr class="row1">
-		<td class="col0"> CoCl2 (µM) </td><td class="col1 leftalign">  </td><td class="col2"> 0 </td><td class="col3"> 100 </td><td class="col4"> 150 </td><td class="col5"> 200 </td><td class="col6"> 0 </td><td class="col7"> 100 </td><td class="col8"> 150 </td><td class="col9"> 200 </td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_510'> </div>
-</div>
-<div class='secedit editbutton_section editbutton_508'> </div>
-<h2 class="sectionedit511" id="section061017">06.10.17</h2>
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_511'> </div>
-<h3 class="sectionedit512" id="sample_preparation_sds-page_and_western_blot3">Sample preparation, SDS-PAGE and Western Blot</h3>
-<div class="level3">
-<p>
-Experiments were performed as described earlier.
-</p>
-<div class="table sectionedit513"><table class="inline">
-	<tr class="row0">
-		<td class="col0"> cell line </td><td class="col1"> ladder </td><td class="col2"> WT </td><td class="col3"> WT </td><td class="col4"> WT </td><td class="col5"> WT </td><td class="col6"> HRE </td><td class="col7"> HRE </td><td class="col8"> HRE </td><td class="col9"> HRE </td>
-	</tr>
-	<tr class="row1">
-		<td class="col0"> CoCl2 (µM) </td><td class="col1 leftalign">  </td><td class="col2"> 0 </td><td class="col3"> 400 </td><td class="col4"> 0 </td><td class="col5"> 400 </td><td class="col6"> 0 </td><td class="col7"> 400 </td><td class="col8"> 0 </td><td class="col9"> 400 </td>
-	</tr>
-	<tr class="row2">
-		<td class="col0"> CMV:HIF </td><td class="col1"> </td><td class="col2"> - </td><td class="col3"> - </td><td class="col4"> + </td><td class="col5"> + </td><td class="col6"> - </td><td class="col7"> - </td><td class="col8"> + </td><td class="col9"> + </td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_513'> </div>
-</div>
-<div class='secedit editbutton_section editbutton_512'> </div>
-<h2 class="sectionedit514" id="section071017">07.10.17</h2>
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_514'> </div>
-<h3 class="sectionedit515" id="t7e1_assay_on_puromycin_selected_non-clonal_jurkat_hif1a-ko">T7E1 assay on puromycin selected non-clonal Jurkat HIF1A-KO</h3>
-<div class="level3">
-<p>
-T7E1 assay was performed according to protocol. PCR for HEK CCRdelta32 positive control and WT/KOJK HIF1A exon 6 (KO1) and exon 5(KO3) were each performed twice. gDNA was extracted according to protocol in cell cluture lab book (16.05.17).<br/>
-</p>
-<ul>
-<li class="level1"><div class="li"> sgRNA1 vs. exon 6 </div>
-</li>
-<li class="level1"><div class="li"> sgRNA3 vs. exon 5</div>
-</li>
-<li class="level1"><div class="li"> WT </div>
-</li>
-<li class="level1"><div class="li"> 3/4 of semi-confluent 6 cm dish for each</div>
-</li>
-</ul>
-<div class="table sectionedit516"><table class="inline">
 	<thead>
 	<tr class="row0">
-		<th class="col0">volume</th><th class="col1">component</th>
+		<th class="col0">CoCl2 (µmol/ml)</th>
 	</tr>
 	</thead>
 	<tr class="row1">
-		<td class="col0"> x </td><td class="col1"> gDNA (300-400 ng)</td>
+		<td class="col0">0</td>
 	</tr>
 	<tr class="row2">
-		<td class="col0"> 10 µl </td><td class="col1"> 5x buffer </td>
+		<td class="col0">50</td>
 	</tr>
 	<tr class="row3">
-		<td class="col0"> 1.5 µl </td><td class="col1"> dNTPs </td>
+		<td class="col0">100</td>
 	</tr>
 	<tr class="row4">
-		<td class="col0"> 1.25 µl </td><td class="col1"> each primer </td>
+		<td class="col0">200</td>
 	</tr>
 	<tr class="row5">
-		<td class="col0"> 0.5 µl </td><td class="col1"> Q5 </td>
+		<td class="col0">400</td>
 	</tr>
 	<tr class="row6">
-		<td class="col0"> 35.5-x µl </td><td class="col1"> H2O </td>
+		<td class="col0">600</td>
 	</tr>
 </table></div>
-<div class='secedit editbutton_table editbutton_516'> </div>
+<!-- EDIT159 TABLE [31114-31165] -->
 <p>
-protocol HIF-KO confirmation<br/>
+<em class="u">Results:</em>
-annealing at 59°C, 40 cycles<br/>
 </p>
+<div class="table sectionedit160"><table class="inline">
-<p>
-Very good concentrations were obtained by PCR clearup (Qiagen), no effect of template concentration on product was observed.<br/>
-</p>
-</div>
-<div class='secedit editbutton_section editbutton_515'> </div>
-<h2 class="sectionedit517" id="section81017">8.10.17</h2>
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_517'> </div>
-<h3 class="sectionedit518" id="annealing_and_t7_assay">Annealing and T7 Assay</h3>
-<div class="level3">
-<ul>
-<li class="level1"><div class="li"> 500 ng per sample in 1x NEB2, each condition double amount annealed (for +/- T7)</div>
-</li>
-<li class="level1"><div class="li"> HEKCCR5delta32 / JK KO1 pure / JK KO1:WT ex 6 1:1 / JK WT ex 6 pure / JK KO3 pure / JK KO3:WT ex 5 1:1 / JK WT ex 5 pure</div>
-</li>
-<li class="level1"><div class="li"> sample volume adjusted to highest sample volume</div>
-</li>
-<li class="level1"><div class="li"> 10 min 95°C in heatblock, closed with parafilm, passive cooldown in heatblock ~ 3 h</div>
-</li>
-</ul>
-<p>
-T7
-</p>
-<ul>
-<li class="level1"><div class="li"> 12 µl annealed DNA per sample mixed on ice with 1.5 µl of 1:1 mix T7E1 and NEB2 2x buffer</div>
-</li>
-<li class="level1"><div class="li"> 20 min at 37°C in PCR cycler</div>
-</li>
-<li class="level1"><div class="li"> stoped with 6x Orange loading dye on ice</div>
-</li>
-<li class="level1"><div class="li"> loaded on 2% agarose EtBr gel with untreated control for each sample</div>
-</li>
-<li class="level1"><div class="li"> run at 95 V for 50 min</div>
-</li>
-<li class="level1"><div class="li"> positive control did not work</div>
-</li>
-<li class="level1"><div class="li"> 500 ng per sample in 1x NEB2</div>
-</li>
-</ul>
-</div>
-<div class='secedit editbutton_section editbutton_518'> </div>
-<h2 class="sectionedit519" id="section111017">11.10.17</h2>
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_519'> </div>
-<h3 class="sectionedit520" id="kryo_stocks_cas9-puro_grna1_and_grna3">Kryo stocks Cas9-puro gRNA1 and gRNA3</h3>
-<div class="level3">
-<p>
-Kryo stocks of puromycin selected, non clonal Jurkat cells were generated according to protocol. From 1 confluent 6 cm dish cryostock was generated by storing cells in 1 ml containing 90% FBS and 10% DMSO. After 2 days at -80°C cells were stored in liquid nitrogen tank.
-</p>
-</div>
-<div class='secedit editbutton_section editbutton_520'> </div>
-<h2 class="sectionedit521" id="section151017">15.10.17</h2>
-<div class="level2">
-</div>
-<div class='secedit editbutton_section editbutton_521'> </div>
-<h3 class="sectionedit522" id="transient_and-gate_test">Transient AND-gate test</h3>
-<div class="level3">
-<p>
--well plates of HEK KD134 cells were transfected using PEI to test AND-gate transiently.
-</p>
-<p>
-PEI transfection protocol
-</p>
-<ol>
-<li class="level1"><div class="li"> seed cell one day before transfection, cell density should be 70-80%</div>
-</li>
-<li class="level1 node"><div class="li"> PEI transfection</div>
-<ol>
-<li class="level2"><div class="li"> mix 3 µg DNA in 100 µl serum-free medium</div>
-</li>
-<li class="level2"><div class="li"> mix 9 µg PEI in 100 µl serum-free medium</div>
-</li>
-<li class="level2"><div class="li"> incubate both tubes 10 min at room temperature</div>
-</li>
-<li class="level2"><div class="li"> add the PEI solution to DNA solution and mix, then incubate 15 min at RT</div>
-</li>
-<li class="level2"><div class="li"> remove medium from cells</div>
-</li>
-<li class="level2"><div class="li"> add 800 µl serum-free medium to DNA/PEI mixture and add to the cells</div>
-</li>
-<li class="level2"><div class="li"> incubate ~3 h at 37°C</div>
-</li>
-<li class="level2"><div class="li"> addd 2 ml culture medium to cells</div>
-</li>
-</ol>
-</li>
-<li class="level1"><div class="li"> Change medium after 3-5 h</div>
-</li>
-</ol>
-<p>
-DNA: 1 µg per plasmid, 3 µg total in each transfection<br/>
-PEI: 3:1 per DNA, i.e. 9 µg per transfection<br/>
-</p>
-<p>
-KD134<br/>
-</p>
-<div class="table sectionedit523"><table class="inline">
 	<thead>
 	<tr class="row0">
-		<th class="col0"> </th><th class="col1">CRE:HIF</th><th class="col2">HRE:LUC</th><th class="col3">pUC</th>
+		<th class="col0">HEK293T</th><th class="col1">Jurkat</th>
 	</tr>
 	</thead>
 	<tr class="row1">
-		<th class="col0">400 µM CoCl2 </th><td class="col1"> pH7.7 </td><td class="col2"> pH7.1 </td><td class="col3"> pH6.1 </td>
+		<td class="col0"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=hek_hre_rfi.jpg" class="media" title="hek_hre_rfi.jpg"><img src="https://static.igem.org/mediawiki/2017/8/86/T%E2%80%93FREIBURG%E2%80%93Modeling-18.10.17-31.jpg" class="mediacenter" alt="" width="400" /></a></td><td class="col1">high mCherry <a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=jk_hre_high_rfi.jpg" class="media" title="jk_hre_high_rfi.jpg"><img src="https://static.igem.org/mediawiki/2017/8/8e/T%E2%80%93FREIBURG%E2%80%93Modeling-18.10.17-32.jpg" class="mediacenter" alt="" width="400" /></a></td>
 	</tr>
 	<tr class="row2">
-		<th class="col0">400 µM CoCl2</th><td class="col1"> pH7.7 + Forskulin + IBMX </td><td class="col2"> pH7.1 + Forskulin + IBMX </td><td class="col3"> pH6.1 + Forskulin + IBMX </td>
+		<td class="col0"> </td><td class="col1">low mCherry <a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=jk_hre_low_rfi.jpg" class="media" title="jk_hre_low_rfi.jpg"><img src="https://static.igem.org/mediawiki/2017/a/a3/T%E2%80%93FREIBURG%E2%80%93Modeling-18.10.17-33.jpg" class="mediacenter" alt="" width="400" /></a></td>
 	</tr>
 </table></div>
-<div class='secedit editbutton_table editbutton_523'> </div>
+<!-- EDIT160 TABLE [31181-31322] -->
-<p>
-KD134<br/>
-</p>
-<div class="table sectionedit524"><table class="inline">
-	<thead>
-	<tr class="row0">
-		<th class="col0"> </th><th class="col1">CRE:HIF CMV:TDAG HRE:LUC </th><th class="col2">CRE:HIF HRE:LUC SV40:TDAG</th><th class="col3">CMV:HIF HRE:LUC pUC</th>
-	</tr>
-	</thead>
-	<tr class="row1">
-		<th class="col0">0 µM CoCl2 </th><td class="col1"> pH6.5 + Forskulin + IBMX </td><td class="col2"> pH6.5 + Forskulin + IBMX </td><td class="col3 leftalign">  </td>
-	</tr>
-	<tr class="row2">
-		<th class="col0">400 µM CoCl2</th><td class="col1"> pH6.5 + Forskulin + IBMX </td><td class="col2"> pH6.5 + Forskulin + IBMX </td><td class="col3 leftalign">  </td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_524'> </div><div class="table sectionedit525"><table class="inline">
-	<tr class="row0">
-		<th class="col0">0 µM CoCl2 </th><td class="col1"> HRE:Luc 1:2 pUC </td><td class="col2 leftalign">  </td><td class="col3 leftalign">  </td>
-	</tr>
-	<tr class="row1">
-		<th class="col0">400 µM CoCl2</th><td class="col1"> HRE:Luc 1:2 pUC </td><td class="col2 leftalign">  </td><td class="col3 leftalign">  </td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_525'> </div>
-<p>
-WT<br/>
-</p>
-<div class="table sectionedit526"><table class="inline">
-	<tr class="row0">
-		<th class="col0">0 µM CoCl2 </th><td class="col1"> HRE:Luc 1:2 pUC </td><td class="col2 leftalign">  </td><td class="col3 leftalign">  </td>
-	</tr>
-	<tr class="row1">
-		<th class="col0">400 µM CoCl2</th><td class="col1"> HRE:Luc 1:2 pUC </td><td class="col2 leftalign">  </td><td class="col3 leftalign">  </td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_526'> </div>
 </div>
-<div class='secedit editbutton_section editbutton_522'> </div>
+<!-- EDIT154 SECTION "input tests in HEK and Jurkat cells" [29543-31322] -->
-<h2 class="sectionedit527" id="section161017">16.10.17</h2>
+<h2 class="sectionedit161" id="section191017">19.10.17</h2>
 <div class="level2">
 </div>
-<div class='secedit editbutton_section editbutton_527'> </div>
+<!-- EDIT161 SECTION "19.10.17" [31323-31343] -->
-<h3 class="sectionedit528" id="induction_of_transient_and-gate_test">Induction of transient AND-gate test</h3>
+<h3 class="sectionedit162" id="hre_ctla4_and_hre_induction">HRE, Ctla4 and HRE induction</h3>
 <div class="level3">
 <p>
-AND-gate was induced 24 h after start of PEI by regulating pH and adding CoCl2 and Forskulin according to schemes above (Forskulin 1:1000 of 100 mM stock, IBMX 1:1000 of 100 mM stock).
+- medium change for Ctla4 and HRE cells: PBS (10% FCS, 0.5% glucose, HEPES)<br/>
-</p>
-</div>
+- medium change for CRE: PBS (10% FCS, 0.5% glucose, HEPES) pH = 7.7<br/>
-<div class='secedit editbutton_section editbutton_528'> </div>
-<h2 class="sectionedit529" id="section171017">17.10.17</h2>
-<div class="level2">
-</div>
+- fluorescence measurement after 0h, 3h, 6h, 12h, 24h, 30h<br/>
-<div class='secedit editbutton_section editbutton_529'> </div>
-<h3 class="sectionedit530" id="change_of_all_media">Change of all media</h3>
-<div class="level3">
-<p>
-All media were changed 12 h after induction, all conditions were added as before to achieve more constant pH induction.
 </p>
-</div>
-<div class='secedit editbutton_section editbutton_530'> </div>
-<h2 class="sectionedit531" id="section181017">18.10.17</h2>
-<div class="level2">
 </div>
-<div class='secedit editbutton_section editbutton_531'> </div>
-<h3 class="sectionedit532" id="sds-page4">SDS-PAGE</h3>
-<div class="level3">
-<p>
-SDS-PAGE was performed as previously.<br/>
-</p>
-<p>
-KD CRE:HIF, HRE:LUC, SV40TDAG
-</p>
-<div class="table sectionedit533"><table class="inline">
-	<tr class="row0">
-		<th class="col0">pH</th><td class="col1"> 6.5 </td><td class="col2"> 7.1 </td><td class="col3"> 7.7 </td><td class="col4"> 6.5 </td><td class="col5"> 7.1 </td><td class="col6"> 7.7 </td>
-	</tr>
-	<tr class="row1">
-		<th class="col0">Ftl </th><td class="col1"> - </td><td class="col2"> - </td><td class="col3"> - </td><td class="col4"> + </td><td class="col5"> + </td><td class="col6"> + </td>
-	</tr>
-</table></div>
-<div class='secedit editbutton_table editbutton_533'> </div><div class="tags"><span>
-	<a href="/igem2017/doku.php?id=tag:labbooks&amp;do=showtag&amp;tag=labbooks" class="wikilink1" title="tag:labbooks" rel="tag">labbooks</a>,
-	<a href="/igem2017/doku.php?id=tag:labor" class="wikilink1" title="tag:labor" rel="tag">labor</a>
-</span></div>
 </div>
-<div class='secedit editbutton_section editbutton_69'> </div>
+</div>
-    <!-- end rendered wiki content -->
+</div>
-    <div class="clearer"></div>
+</div>
-  </div>
-  <!-- end div id bodyContent -->
+    </div>
+<!-- end div id=content -->
 </div>
@@ Line 7,286: / Line 2,794: @@
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 </div>
-  <script>
+<script>
+function openModal() {
+  document.getElementById('myModal').style.display = "block";
+}
+function closeModal() {
+  document.getElementById('myModal').style.display = "none";
+}
+var slideIndex = 1;
+showSlides(slideIndex);
+function plusSlides(n) {
+  showSlides(slideIndex += n);
+}
+function currentSlide(n) {
+  showSlides(slideIndex = n);
+}
+function showSlides(n) {
+  var i;
+  var slides = document.getElementsByClassName("mySlides");
+  var dots = document.getElementsByClassName("demo");
+  var captionText = document.getElementById("caption");
+  if (n > slides.length) {slideIndex = 1}
+  if (n < 1) {slideIndex = slides.length}
+  for (i = 0; i < slides.length; i++) {
+      slides[i].style.display = "none";
+  }
+  for (i = 0; i < dots.length; i++) {
+      dots[i].className = dots[i].className.replace(" active", "");
+  }
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+}
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@@ Line 7,305: / Line 2,856: @@
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+</script>
-</div>
-</div>
-</div>
-</div>
-</div>
-<!-- end footer -->
-<img src="/igem2017/lib/exe/indexer.php?id=labor%3Acell_culture&amp;1508332270" width="2" height="1" alt="" />
 </body>
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 {{Freiburg/Footer}}

Difference between revisions of "Team:Freiburg/Notebook Knockout"

Revision as of 17:54, 23 October 2017

Lab Notebook Modeling

06.06.17

Titration RPMI 1640 vs. lactic acid

13.06.17

Cell survival and GFP-stability at different pH-values

14.06.17

pH-measurement of used medium (13.06.17)

17.06.17

Investigation of the pH-changes

19.06.17

pH change vs. incubation time

04.07.17

VEGF dilution

06.07.17

pCTLA4 vs. [VEGF]

18.07.17

PEI transfection of plasmids pIG17_022/23/37/86

19.07.17

VEGF test

05.08.17

Interlab Study transformation

Interlab Study OD600 calibration measurements

Interlab Study fluorescein standard curve measurements

06.08.17

pick colonies from Interlab Study

07.08.17

Interlab Study cell measurements

19.08.-27.08.17

pH change over time in incubator with RPMI1640 and acid

10.08.17

Transformation of pWHE-644

12.08.17

Miniprep of pWHE-644 in DH3-α

Test digest of pWHE-644

13.08.17

Miniprep of CMV-VEGFR2 (pIG_138)

Test digest of CMV-VEGFR2 (pIG_138)

13.08.17

Test digest of CMV-VEGFR2 (pIG_138)

16.08.17

pH test with Jurkat cells

16.08.17

Test digest of CMV-VEGFR2 (pIG_138)

Ctla4 induction

17.08.17

PEI transfection of HEK cells

Flow cytometry

18.08.17

BioRad electroporation

Calcium test

20.08.17

Biorad electroporation

pH tests with Jurkat

Miniprep of pIG_034

21.08.17

sequencing of pIG_138

22.08.17

Miniprep of pIG_017, pIG_031, pIG_034, pIG_037

up-concentration of pIG_017, pIG_031, pIG_034, pIG_037

25.08.17

Miniprep of pIG_017, pIG_037

07.09.17

PEI transfection of HEK cells

Gel extraction of M1-receptor without CFP

08.09.17

Colony PCR of M1

20.08.17

pCRE induction

10.09.17

PEI transfection of plasmids pIG17_022/23/37/86

HRE induction

Repetition of the Interlab Study

PEI transfection of HEK cells

16.09.17

Pick colonies of Interlab Study

pCRE induction in HEK cells

17.09.17

PEI transfection of plasmid pIG17_031