Revision as of 18:32, 20 October 2017

labor:cell_culture - iGEM 2017

Lab Notebook Cell Culture

PEI transfection of HEK cells

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 (2.5µg from each plasmid)
Add 15 µl PEI(1µg/µl) to the mix while vortexing (PEI:DNA=3:1)
incubate 15 min at RT
Add the mix to the culture

13.05.17

PEI transfection of HEK, Jurkat, HPB-ALL and HUT78 cells

Split cells 1:5 to a 6-well plate: 100 µl/well (on 12.05.17)
For each sample: 3 µg plasmid DNA + 200 µl serum free DMEM
Establish Mastermix
Add 9 µg PEI to each mix while vortexing (PEI:DNA=3:1)
15 min incubation
Add the Mix to the cells: 275.25 µl GFP-Mix or 215.5 µl to corresponding samples
After 48h: Fluorescence Microscopy

Plasmid number	component	concentration[ng/µL]	volume[µL] for 12 µg
pIG17_008	CMV_mCherry-delta-PstI_pA	471.5	25
pIG17_009	CMV_EGFP_pA	368.4	30

23.05.17

PEI transfection of HEK, Jurkat, HPB-ALL and HUT78 cells

Plasmid number	component
pIG17_008	CMV_mCherry-delta-PstI_pA
pIG17_009	CMV_EGFP_pA

Split cells 1:5 to a 6-well plate: 330 µl (from 10 cm plate)/well (22.05.17)
For each sample: 3 µg plasmid DNA + 200 µl serum free DMEM
Establish Mastermix
Add PEI to each mix while vortexing (PEI:DNA=3:1)
15 min incubation
Add the Mix to the cells
After 48h: Fluorescence Microscopy & Flow Cytometry

Lipotransfection of Jurkat, HPB-ALL and HUT78 cells

Plasmid number	component
pIG17_009	CMV_EGFP_pA

pGFP: 2.5 µg/well
Mix I: 150 µl serum free DMEM and 6 µl LTX reagent
Mix II: 700 µl SERUM FREE DMEM + 2.5 µg pGFP + 14 µl PLUS reagent
Mix 150 µl Mix I with 150 µl Mix II
5 min incubation at RT
Add 300 µl to cells

30.05.17

BioRad electroporation

Cell lines: HPB-ALL, HUT78 and Jurkat Plasmid: pIG17_009 (GFP plasmid)

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 3 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

preparation for lentiviral transduction

Plate 4 mio. Hek cells in 10 cm plate. (Will be around 7-8 mio cell the next day)
(optional: 5-6 mio. Hek cells in 15 cm plate)

06.06.17

Production of Lentivirus

Transfection of HEK cells for virus production.

Lentiviral packaging cells	Hek293T cells
Plate size	10 cm
Total DNA/plate	12.87 µg
Transfer: gag/pol: env ratio	2:1:1
Construct	6.43 µg
pCMV∆R8.74	3.22 µg
pMD2G	3.22 µg
N/P ration	15

Plasmid number	component	concentration[ng/µL]	volume [µL]
pIG17_082	Transfer plasmid with GFP p526	253	25.4
pIG17_003	Envelope plasmid pMD2G	105	30.7
pIG17_004	Packaging Plasmid pCMV∆R8.74	150	21.5

Packaging Mastermix

Component	1x [µL]	2.1x [µL]
pIG17_003	30.63	45.03
pIG17_004	21.44	64.33
150mM NaCl	441.49	927.12
total	493.56	1036.476

PEI Mastermix

Component	1x [µL]	2.1x [µL]
23.2mM (1 µg/µl)PEI	24.94	52.41
150mM NaCl	475.04	997.59
total	499.98	1050

07.06.17

Amaxa electroporation

Cell lines: HUT78 and Jurkat Plasmid: pIG17_008 (mCherry plasmid)

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 3 µg pIG17_008
Transfer the mixture into cuvette
Amaxa Programm: X-005
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

Production of Lentivirus

Collect the medium (containing virus supernatant) and add new medium after 24h

BioRad electroporation

Cell lines: HUT78 and Jurkat Plasmid: pIG17_009 (GFP plasmid)

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 3 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

08.06.17

Production of Lentivirus

Collect the medium (containing virus supernatant) and add new medium after 48h
Mix with medium collected after 24h

Flow Cytometry

negative controls: Hut78 and Jurkat cells.
positive control: GFP_Jurkat(p526)
Amaxa Samples: Hut78 (mCherry and GFP) and Jurkat (mCherry)
BioRad Samples: Hut78 (GFP) and Jurkat (GFP)

Count the cells
resuspend cell pellet (about 1 mio.) with FACS Buffer (PBS with 1%FCS)

09.06.17

Lentiviral transduction

Plate about 200000 cells for each wells.
Incubate the plates over weekend in incubator.

Jurkat sample	Medium [ml]	Virus [ml]
1	1	1
2	0	2

Hut78 sample	Medium [ml]	Virus [ml]
1	2	0
2	1.5	0.5
3	1	1
4	0	2

HEK sample	Medium [ml]	Virus [ml]
1	2	0
2	1.5	0.5
3	1	1
4	0	2

10.06.17

BioRad electroporation

Cell lines: Hut78
Plasmid: pIG17_008 (mCherry plasmid)

Component	Amount
Hut cells	3 mio. cells
pIG17_008	5 µg

Setting	Capacitance[µF]	Voltage	Cuvette size[cm]	Time Constance [msec]
Setting 1	250	150	0.2	4.0
Setting 2	500	120	0.2	10.1

12.06.17

Lentiviral transduction

After 72h → Change the medium of the plates (for suspension cells: first centrifuge and then resuspend cell pellet with new medium)
Incubation over night for FACS

Flow Cytometry for BioRad electroporation on June 10th

no positive population

13.06.17

Flow Cytometry for lentiviral infected cells

with ration of 1:1 (Medium vs. Virus medium) cells were most frequently infected and viable.

BioRad Electroporation

Cell lines: Jurkat & Hut78
Plasmid: pIG17_009
Cell count: 1 mio. (Jurkat) 4 mio. (Hut78)
Settings:

Cell type	Setting	Capacitance[µF]	Voltage	resistence	Time Constance [msec]
Jurkat	Setting 1	300	250	∞	35
Jurkat	Setting 2	350	200	1000Ω	35
Hut78	Setting 1	250	150	∞	50
Hut78	Setting 2	500	120	∞	50

14.06.17

Amaxa electroporation

Cell lines: Jurkat & Hut78
Plasmid: pIG17_009
Cell count: 1 mio.
Program: X-005
Flow cytometry after 24h.

Flow Cytometry for BioRad electroporated JK cells

No positive cell population.

15.06.17

Flow Cytometry for BioRad electroporated Hut78 cells and Amaxa electroporated T cells

no positive cell population.

16.06.17

Mycoplasma test(lentiviral transduced Jurkat and Hut78)

Jurkat(p526): mycoplasma positive
Hut78(p526):mycoplasma positive
HEK(p526): mycoplasma negative

No cell sorting, stock cultures should be sent for test.

17.06.17

BioRad Electroporation with JK cells

Cell lines: Jurkat
Plasmid: pIG17_009
Cell count: 2 mio./ approach
4 approaches:

Approach	Capacitance[µF]	Voltage	Cuvette size[cm]	Time Constance [msec]
1:negative control	960	250	0.2	35
2:condition 1	960	250	0.2	35
3:condition 2	960	250	0.2	35
4:condition 3	960	250	0.2	40

PEI transfection with HEK cells

4 approaches:

1 transfection with pIG17_009
2 transfections with pIG17_013
1 untransfected as control

Split cells 1:5 to a 6-well plate: 100 µl/well (on 16.06.17)
For each sample: 3 µg plasmid DNA + 200 µl serum free DMEM
Establish Mastermix
Add 9 µg PEI to each mix while vortexing (PEI:DNA=3:1)
15 min incubation
Add the Mix to the cells
After 48h: Fluorescence Microscopy & Flow cytometry

Plasmid number	component
pIG17_013	pHRE_Ptal_EGFP_pA

19.06.17

Hypoxia test (CoCl2)

Cell: via PEI transfected HEK cells.
CoCl2 stock: 1M
End concentration of CoCl2: 100µM
Cell observation under fluorescence microscopy after 2h:

-By adding CoCl2, the HRE-GFP transfected cells showed less fluorescence signal than without CoCl2…

Flow cytometry: no expected signal

BioRad Electroporation

Cell lines: Jurkat
Plasmid: pIG17_009
Cell count: 2 mio./ approach
Setting: 96 well plates, well set 1(ABCD1)
4 approaches:

Approach	Capacitance[µF]	Voltage	Cuvette size[cm]	Time Constance [msec]
1:negative control	960	250	0.2	35
2:condition1	960	250	0.2	35
3:condition2	960	250	0.2	30
4:condition3	960	250	0.2	40

20.06.17

BioRad electroporation

Cell lines: Jurkat
Plasmid: pIG17_009
Cell count: 2 mio./ approach
Setting: 96 well plates, well set 1(ABCD1), Expotential
4 approaches:

Approach	Capacitance[µF]	Voltage	Cuvette size[cm]	Resistance
1:negative control	950	250	0.2	1000Ω
2:condition1	350	250	0.2	∞
3:condition2	300	300	0.2	1000Ω
4:condition3	300	250	0.2	1000Ω

21.06.17

Mycoplasma-Test (Stock cultures)

HEK293T: mycoplasma negative
Jurkat: mycoplasma negative
Hut78: mycoplasma positive → abort culturing of Hut78!

Flow Cytometry (BioRad on Jun.19th)

Not successful

24.06.17

PEI transfection (pIG17_013) for hypoxia test

In total: 3*6 wells
Transfection with pIG17_009 as positive control: 1 well/plate
Transfection with pIG17_013: 4 well/plate

Split the cells 1:5 into 10 cm plate (on 23.06.17)
Thaw PEI reagent at RT
Dilute plasmid in 1 ml serum free DMEM (3µg from each plasmid)
Add 15 µl PEI(1µg/µl) to the mix while vortexing (PEI:DNA=3:1)
incubate 15 min at RT
Add 200µl mix to wells

25.06.17

Hypoxia test (CoCl2)

Cell: 1 plate of via PEI transfected HEK cells. (from 24.06)
CoCl2 stock: 1M
End concentration of CoCl2: 100µM

Well	End concentration of CoCl2[µM]	Volume of Stock CoCl2[µl]
1: untransfected	100	200
2:pIG17_009 transfected	-	-
3:pIG17_013 transfected	-	-
4:pIG17_013 transfected	50	100
5:pIG17_013 transfected	100	200
6:pIG17_013 transfected	200	400

* Cell observation under fluorescence microscopy every hour after CoCl2 treatment

Flow cytometry after 8 hours

BioRad Electroporation

Cell lines: Jurkat
Plasmid: pIG17_009
Cell count: 2 mio./ approach
Setting: 12 well plates, well set 1
3 approaches:

Approach	Capacitance[µF]	Voltage	Resistance	Time Constance [msec]
1:3 µg plasmid	960	250	∞	20
2:6 µg plasmid	960	250	∞	20
3:9 µg plasmid	960	250	∞	20

Flow cytometry after 24h.

26.06.17

Hypoxia test (CoCl2)

1.Plate

Duration of Treatment	Well	End concentration of CoCl2[µM]	Volume of Stock CoCl2[µl]
6h	1: untransfected	100	200
6h	2:pIG17_009 transfected	-	-
6h	3:pIG17_013 transfected	-	-
6h	4:pIG17_013 transfected	50	100
6h	5:pIG17_013 transfected	100	200
6h	6:pIG17_013 transfected	200	400

2.Plate

Duration of Treatment	Well	End concentration of CoCl2[µM]	Volume of Stock CoCl2[µl]
24h	1: untransfected	100	200
24h	2:pIG17_009 transfected	-	-
24h	3:pIG17_013 transfected	-	-
24h	4:pIG17_013 transfected	50	100
24h	5:pIG17_013 transfected	100	200
24h	6:pIG17_013 transfected	200	400

Flow Cytometry (Hypoxia test & BioRad)

Hypoxia test

No induction of GFP expression under hypoxia condition Problems:

Cells stayed attached to the ground of plates even after treating with trypsin
Cells clumps instead of single cell were detected

BioRad Electroporation

By increasing the amount of plasmid DNA, some cells were potentially transfected!!!

27.06.17

BioRad Electroporation

9µg for each approach

Setting: 12 well plate, 1 well set, 1 pulse

GFP Plasmid: pIG17_009

Approach	Capacitance[µF]	Voltage	Resistance	Time Constance [msec]
1	950	250	∞	20
2	950	250	1000Ω	20
3	950	250	1500Ω	20

Knock out

Approach	Plasmid	Capacitance[µF]	Voltage	Resistance	Time Constance [msec]
1	203 (Cas9) + 104 (gRNA)	950	250	∞	20
2	203 (Cas9)+ scramble	950	250	∞	20
3	sgRNA3 (gRNA_GFP)	950	250	∞	20
4	sgRNA3 (gRNA_GFP)	950	250	∞	20

28.06.17

Flow Cytometry: electroporation on June 27th

29.06.17

Production of Lentivirus

Transfection of HEK cells for virus production.

Lentiviral packaging cells	Hek293T cells
Plate size	10 cm
Total DNA/plate	12.87 µg
Transfer: gag/pol: env ratio	2:1:1
Construct	6.43 µg
pCMV∆R8.74	3.22 µg
pMD2G	3.22 µg
N/P ration	15

Plasmid number	component	concentration[ng/µL]	volume [µL]
pIG17_?	LentiCRISPR(AG Cathomen)	1100	5.85
pIG17_003	Envelope plasmid pMD2G	68	47.4
pIG17_004	Packaging Plasmid pCMV∆R8.74	209	15.4

Packaging Mastermix

Component	1x [µL]	2.1x [µL]
pIG17_003	47.4	99.54
pIG17_004	15.4	32.34
150mM NaCl	441.49	927.12
total	504.3	1059

PEI Mastermix

Component	1x [µL]	2.1x [µL]
23.2mM (1µg/µl)PEI	24.94	52.41
150mM NaCl	475.04	997.59
total	499.98	1050

30.06.17

Production of Lentivirus

Collect the medium (containing virus supernatant) and add new medium after 24h

01.07.17

Production of Lentivirus

Collect the medium (containing virus supernatant) and add new medium after 24h
Mix the virus medium (24h + 48h)

Lentiviral transduction

Plate about 200000 cells for each wells.
Incubate the plates over weekend in incubator.

Jurkat sample	Medium [ml]	Virus [ml]
1	2	0
2	1.5	0.5
3	1	1
4	0.5	1.5
5	0	2

PEI transfection with HEK for Hypoxia Test

Prepare 2*6-well Plates of HEK cells
Transfect cells via PEI:

well	plasmid
1	-
2	pIG17_009
3	pIG17_013
4	pIG17_013
5	pIG17_013
6	pIG17_013

BioRad Electroporation: Repeat pGFP transfection

9µg for each approach

Setting: 12 well plate, 1 well set, 1 pulse

Approach	Capacitance[µF]	Voltage	Resistance	Time Constance [msec]
1: no plasmid	950	250	∞	20
2: Mock plasmid	950	250	∞	20
3: pIG17_009	950	250	∞	20
4: pIG17_009	950	250	1000Ω	20
5: pIG17_009	950	250	1500Ω	20

02.07.17

CoCl2 treatment with Jurkat cells

Cells treated with CoCl2 built clumps, which depends on the incubation time and CoCl2 concentrations.

Repeat Hypoxia test (24h after PEI)

No induction of GFP expression under hypoxia condition

Add different concentrations of CoCl2 (50µM, 100µM and 200 µM)
Observe the cells under fluorescence microscope 3h, 6h, 8h, 16h, 18h and 24h after CoCl2 treatment
No detectable induction of gene expression for every approach

Flow Cytometry (BioRad on July 1st)

Both negative and positive controls showed abnormal populations → need to repeat the experiments.

03.07.17

Repeat Hypoxia test (48h after PEI)

No induction of GFP expression under hypoxia condition

Add different concentrations of CoCl2 (50µM, 100µM and 200 µM)
Observe the cells under fluorescence microscope 3h, 6h, 8h, 16h, 18h and 24h after CoCl2 treatment
No detectable induction of gene expression for every approach

04.07.17

Lentiviral transduction (pLenti-CRISPR from AG Cathomen)

Change the medium and add puromycin for selection

BioRad Electroporation: Repeat pGFP transfection

9µg for each approach

Setting: 12 well plate, 1 well set, 1 pulse

Approach	Capacitance[µF]	Voltage	Resistance	Time Constance [msec]
1: no plasmid	950	250	∞	20
2: Mock plasmid	950	250	∞	20
3: pIG17_009	950	250	∞	20
4: pIG17_009	950	250	500Ω	20
5: pIG17_082 (p526)	950	250	∞	20

05.07.17

PEI transfection (pIG17_022 and pIG17_023) for CTLA-4 promoter test

In total: 3*6 wells
Transfection with pIG17_009 as positive control: 1 well
Transfection with pIG17_022: 4 wellS
Transfection with pIG17_023: 4 wellS
Per Approach: 3µg DNA, 200µl serum free DMEM and 9µg PEI

Split the cells 1:5 into 10 cm plate (on 03.07.17)
Thaw PEI reagent at RT
Dilute plasmid in 200µl serum free DMEM (3µg from each plasmid)
Add PEI(1µg/µl) to the mix while vortexing (PEI:DNA=3:1)
incubate 15 min at RT
Add 200µl mix to wells

Flow Cytometry

Cells transfected with pIG17_082 showed high transfection efficiency
Cells transfected with our stock pIG17_009 showed low efficiency and low survival rate

06.07.17

Prepare cells for lentivirus-production

Plate about 4 mio. cells, make replicate

VEGF induction with Modeling group

No significant induction

07.07.17

Lentivirus(pIG17_119) production in HEK cells

Transfection of HEK cells for virus production.

Lentiviral packaging cells	Hek293T cells
Plate size	10 cm
Total DNA/plate	12.87 µg
Transfer: gag/pol: env ratio	2:1:1
Construct	6.43 µg
pCMV∆R8.74	3.22 µg
pMD2G	3.22 µg
N/P ration	15

Plasmid number	component	concentration[ng/µL]	volume [µL]
pIG17_119	pCRE_GFP_CMV_mCherry	167	38.5
pIG17_003	Envelope plasmid pMD2G	243.4	13.3
pIG17_004	Packaging Plasmid pCMV∆R8.74	819	4

Packaging Mastermix

Component	1x [µL]	2.1x [µL]
pIG17_003	13.3	28
pIG17_004	4	8.4
150mM NaCl	441.49	927.12
total	458.8	963.52

PEI Mastermix

Component	1x [µL]	2.1x [µL]
23.2mM (1µg/µl)PEI	24.94	52.41
150mM NaCl	475.04	997.59
total	499.98	1050

08.07.17

Lentivirus(pIG17_119) production in HEK cells

Change the medium

BioRad electroporation

4 Approaches (9µg/Approach) Setting: 12 well plate, 1 well set, 1 Pulse, ∞ Resistance

Electroporated without plasmid
CMV-GFP from Nicole (700 ng/µl)
CMV-GFP (Yael, 06.06.17, 140.4 ng/µl)
CMV-GFP (Dennis, 27.06.17, Dennis, 86.8 ng/µl)

09.07.17

Lentivirus(pIG17_119) production in HEK cells

Collect virus medium
Add new medium

10.07.17

Lentivirus(pIG17_119) production in HEK cells

Collect virus medium
Add new medium

11.07.17

PEI transfection (CTLA-4 Test)

In total: 3*6 wells
Transfection with pIG17_009 as positive control: 1 well (plasmid from Nicole); 1 well (Culture from Nicole, from us preped using promega miniprep)
Transfection with pIG17_022: 5 wellS
Transfection with pIG17_023: 5 wellS
Per Approach: 3µg DNA, 200µl serum free DMEM and 9µg PEI

Split the cells 1:5 into 10 cm plate (on 03.07.17)
Thaw PEI reagent at RT
Dilute plasmid in 200µl serum free DMEM (3µg from each plasmid)
Add PEI(1µg/µl) to the mix while vortexing (PEI:DNA=3:1)
incubate 15 min at RT
Add 200µl mix to wells

Lentiviral transduction (pIG17_119) of Jurkat cells

Use 6-well plate
plate 200000 cells/well
Make Titer test with virus medium

The cells were not successfully infected.

12.07.17

BioRad electroporation

Approach	plasmid	Capacitance[µF]	Voltage	Resistance	Time Constance [msec]
1	no plasmid	950	250	∞	20
2	CMV-GFP (Nicole)	950	250	∞	20
3	CMV-GFP (Culture from Nicole, promega mini)	950	250	∞	20
4	pIG17_104: Cas9-GFP-gRNA3	950	250	∞	20

14.07.17

Lentivirus(pIG17_121) production in HEK cells

Transfection of HEK cells for virus production.

Lentiviral packaging cells	Hek293T cells
Plate size	10 cm
Total DNA/plate	12.87 µg
Transfer: gag/pol: env ratio	2:1:1
Construct	6.43 µg
pCMV∆R8.74	3.22 µg
pMD2G	3.22 µg
N/P ration	15

Plasmid number	component	concentration[ng/µL]	volume [µL]
pIG17_121	pCTLA4(380)_GFP_CMV_mCherry	1682	3.82
pIG17_003	Envelope plasmid pMD2G	321.9	10
pIG17_004	Packaging Plasmid pCMV∆R8.74	819	4

Packaging Mastermix

Component	1x [µL]	2.1x [µL]
pIG17_003	10	21
pIG17_004	4	8.4
150mM NaCl	441.49	927.12
pIG17_121	3.82	8
total	504.95	1060

PEI Mastermix

Component	1x [µL]	2.1x [µL]
23.2mM (1µg/µl)PEI	24.94	52.41
150mM NaCl	475.04	997.59
total	499.98	1050

BioRad electroporation

Approach	plasmid	Capacitance[µF]	Voltage	Resistance	Time Constance [msec]
1	no plasmid	950	250	∞	20
2	6µg Cas9-GFP-gRNA3	950	250	∞	20
3	9µg Cas9-GFP-gRNA3	950	250	∞	20
4	12µg Cas9-GFP-gRNA3	950	250	∞	20

15.07.17

collect virus (pIG17_121)

flow cytometry (BioRad 7/14)

16.07.17

PEI transfection

In total: 3*6 wells
One well with non-transfected cells
Transfection with CMV-GFP as positive control: 1 well (JetStar); 1 well (Promega Mini)
Transfection with pIG17_013: 4 wellS
Per Approach: 3µg DNA, 200µl serum free DMEM and 9µg PEI

Split the cells 1:5 into 6-well plate (on 15.07.17)
Thaw PEI reagent at RT
Dilute plasmid in 200µl serum free DMEM (3µg from each plasmid)
Add PEI(1µg/µl) to the mix while vortexing (PEI:DNA=3:1)
incubate 15 min at RT
Add 200µl mix to wells

BioRad electroporation

Approach	plasmid	Capacitance[µF]	Voltage	Resistance	Time Constance [msec]
1	no plasmid	950	250	∞	20
2	CMV-GFP JetStar	950	250	∞	20
3	CMV-GFP promega mini	950	250	∞	20
4	CMV-mCherry (Nicole)	950	250	∞	20
5	pIG17_008 (Jana)	950	250	∞	20

17.07.17

Hypoxia Test in hypoxia incubator (AG Cathomen)

with 1%O2

Flow Cytometry

BioRad didn't work

18.07.17

Hypoxia Test in hypoxia incubator (AG Cathomen)

Fluorescence microscopy
After 24h incubation: cells showed normal morphology and there was no autofluorescence.

Lentiviral transduction

wash the virus-transducted cells (pIG17_119)

BioRad electroporation

Approach	plasmid	Capacitance[µF]	Voltage	Resistance	Time Constance [msec]
1	pIG17_119	950	250	∞	20
2	pIG17_121	950	250	∞	20
3	pIG17_031	950	250	∞	20
4	pIG17_034	950	250	∞	20
5	pIG17_037	950	250	∞	20
5	pIG17_086	950	250	∞	20

PEI transfection (CTLA-4 Test)

In total: 2*12 wells
One well with non-transfected cells
One well: Transfection with CMV-GFP as positive control
Transfection with pIG17_022: 5 wellS
Transfection with pIG17_023: 5 wellS
Transfection with pIG17_037: 5 wellS
Transfection with pIG17_086: 5 wellS
Per Approach: 1.5µg DNA, 100µl serum free DMEM and 4.5µg PEI

Split the cells 1:10 into 12-well plate (on 17.07.17)
Thaw PEI reagent at RT
Dilute plasmid in 100µl serum free DMEM (3µg from each plasmid)
Add PEI(1µg/µl) to the mix while vortexing (PEI:DNA=3:1)
incubate 15 min at RT
Add 100 µl mix to wells

PEI transfection (hypoxia test with CoCl2)

In total: 4*6-well plates
One well in each plate: with non-transfected cells
One well in each plate: Transfection with CMV-GFP as positive control
Transfection with pIG17_013: 8 wellS
Transfection with pIG17_031: 8 wellS

Split the cells 1:10 into 6-well plate (on 17.07.17)
Thaw PEI reagent at RT
Dilute plasmid in 100µl serum free DMEM (3µg from each plasmid)
Add PEI(1µg/µl) to the mix while vortexing (PEI:DNA=3:1)
incubate 15 min at RT
Add 200 µl mix to wells

19.07.17

Concentrate the viruses (pIG17_119 and pIG17_121)

for 20 ml virus medium, use 5ml 20% sucrose
carefully add virus medium to sucrose (very tricky)
centrifuge at 4000 rpm over night
After centrifugation: remove the medium, put the falcon tube upside down and air dry.
slowly add 100 µl PBS (20-30x, also very tricky)
Incubate the pellet for 30-60 min
resuspend the cells 2x
aliquot the virus
store in -80°C freezer

Lentiviral transduction

plate 200000 Jurkat cells/ well in one 6-well plate
add the concentrated virus (pIG17_119 and pIG17_121) (30µl and 50µl)

CoCl2 treatment (24h after PEI)

Treatment with 2*6 well plate: 1 with pIG17_013 and 1 with pIG17_031
Make new 2M CoCl2 stock
add CoCl2 to the cells, end-concentration: 0, 300µM, 500µM and 100µM
Incubation for 24h and track the induction of cell expression

Hypoxia Test in hypoxia incubator (AG Cathomen)

Fluorescence microscopy
After 48h incubation: cells were mostly dead showing autofluorescence.

Flow Cytometry (BioRad on July 20th)

For mCherry excitation: FL3!!!

20.07.17

CoCl2 treatment (48h after PEI)

Treatment with 2*6 well plate: 1 with pIG17_013 and 1 with pIG17_031
Make new 2M CoCl2 stock
add CoCl2 to the cells, end-concentration: 0, 300µM, 500µM and 100µM
Incubation for 24h and track the induction of cell expression

Hypoxia Test in hypoxia incubator (AG Cathomen)

Fluorescence microscopy
After 72h incubation: cells were mostly dead showing autofluorescence.

21.07.17

BioRad Electroporation (Knock-Out plasmids)

Approach	plasmid	Capacitance[µF]	Voltage	Resistance	Time Constance [msec]
1	-	950	250	∞	20
2	CMV-GFP (#503 from Nicole)	950	250	∞	20
3	pIG17_082 (p526)	950	250	∞	20
4	Cas9-GFP-gRNA5	950	250	∞	20
5	Cas9-GFP-scramble	950	250	∞	20

22.07.17

Lentivirus(pIG17_119 and pIG17_121) production in HEK cells

PEI Transfection of HEK cells for virus production.

Lentiviral packaging cells	Hek293T cells
Plate size	10 cm
Total DNA/plate	12.87 µg
Transfer: gag/pol: env ratio	2:1:1
Construct	6.43 µg
pCMV∆R8.74	3.22 µg
pMD2G	3.22 µg
N/P ration	15

Plasmid number	component	concentration[ng/µL]	volume [µL]
pIG17_119	pCRE_GFP_CMV_mCherry	167	38
pIG17_121	pCTLA4(380)_GFP_CMV_mCherry	1682	3.82
pIG17_003	Envelope plasmid pMD2G	105	30.7
pIG17_004	Packaging Plasmid pCMV∆R8.74	208	15.5

Packaging Mastermix

Component	1x [µL]	2.1x [µL]
pIG17_003	30.7	64.5
pIG17_004	15.5	32.6
150mM NaCl	441.49	927.12

PEI Mastermix

Component	1x [µL]	2.1x [µL]
23.2mM (1µg/µl)PEI	24.94	52.41
150mM NaCl	475.04	997.59
total	499.98	1050

22.07.17

Flow Cytometry: BioRad Electroporation 7/21

there was GFP positive signals during flow cytometry measurement but the cells were mycoplasma positive

Lentivirus(pIG17_119 and pIG17_121) production in HEK cells

Collect virus

23.07.17

Lentivirus(pIG17_119 and pIG17_121) production in HEK cells

Collect virus

24.07.17

Mycoplamsa Test

Both Cas9-GFP-gRNA5 and Cas9-GFP-scramble transfected cells were mycoplasma positive

25.07.17

PEI transfection of CHO cells: Hypoxia & Tet system

Split the cells 1:2 in 12-well plate (on 24.07.17)
Per Approach: 1µg DNA + 8µg PEI and fill up to 100 µl with serum free DMEM
Thaw PEI reagent at RT
Add PEI(1µg/µl) to the mix while vortexing
incubate 15 min at RT
Add the mix to cells

Plasmid	Approach	DNA Concentration[ng/µl]	DNA volume	Volume of DMEM[µl]
CMV-GFP (#503 from Nicole)	1	25.5	40	52
CMV-mCherry (AG Hiltbrunner)	1	1200	1	91
pIG17_012	6	253	24	528
pIG17_013	6	583.3	10.3	541.7
pIG17_031	6	746.9	8	544
Tet-GFP (Shima)	2 (protocol from Shima)	520	3.8	180.2
Tet-GFP (Shima)	2 (our protocol)	520	5.8	200

Tetracycline induction of CHO

3h after PEI transfection: remove media, add tetracycline to new media (1:1000 dilution)
Tetracycline Stock: 2mg/ml (End: 1µg per well of 12-well plate)
3h after adding tetracycline: no induction
incubation till the next day: microscopic observation
Gene expression was induced by tetracycline compared to the tetracycline-untreated cells

PEI transfection of HEK cells: Hypoxia

Split the cells 1:5 in 12-well plate (on 24.07.17)
Per Approach: 1.5µg DNA + 4.5µg PEI and 100 µl serum free DMEM
Thaw PEI reagent at RT
Add PEI(1µg/µl) to the mix while vortexing
incubate 15 min at RT
Add the mix to cells

Plasmid	Approach	DNA Concentration[ng/µl]	DNA volume
CMV-GFP (#503 from Nicole)	1	25.5	59
CMV-mCherry (AG Hiltbrunner)	1	1200	1.25
pIG17_012	6	131.4	12
pIG17_013	6	583.3	15.5
pIG17_031	6	746.9	12

26.07.17

CoCl2 treatment

Add corresponding concentration of CoCl2 to (hypoxic plasmid) transfected cells (CHO and HEK)
CoCl2 stock: 2M

Endconcentration of CoCl2[µM]	Volume from Stock[µl]
0	0
100	0.1
200	0.2
300	0.3
500	0.5
1000	1

27.07.17

SEAP assay with HEK cells

- gather 200 µl of cell supernatant
- incubate cell supernatant at 65°C for 30min
- centrifuge cell supernatant for 1 min at 1250g
- add 150 µl supernatant into a cuvette
- add 500 µl 2x SEAP buffer
- add 100 µl pNPP and remove bubbles carefully
- measure in a nano drop every 5min for 2h

Replace Stock cultures with new culture from Toolbox

31.07.17

Mycoplasma Test: New HEK stock

New HEK cells were mycoplasma negative

01.08.17

BioRad Electroporation

Per Approach: 9µg DNA, 2 mio.JK cells

Approach	plasmid	Capacitance[µF]	Voltage	Resistance	Time Constance [msec]
1	pIG17_009 (06.06 Yael)	950	250	∞	20
2	Cas9-GFP-gRNA5	950	250	∞	20
3	Cas9-GFP-scramble	950	250	∞	20
4	KO-Kit: 203+101	950	250	∞	20
5	KO-Kit: 203+104	950	250	∞	20
6	KO-Kit scramble	950	250	∞	20

PEI transfection: Hypoxia - SEAP assay in HEK cells

Per Approach: 1.5µg DNA, 4.5µg PEI, 100µl DMEM

Plasmid number	component	concentrations of the used tubes[ng/µL]	volumes from the used tubes[µL] for 36µg
pIG17_013	HRE-SEAP	583.3 and 88.1	55.7 and 40
pIG17_002	CMV-SEAP	92.0 and 91.4	350 and 41.6

Due to the concentrations and volumes in the tubes for each approach two were used. Per construct 24 wells were used. Each well will have different CoCl2 concentrations. For each approach triplicates will be analysed.

02.08.17

Flow Cytometry of BioRad Electroporation (8/1): KO plasmids

For the knockout Kit we could see the most living GFP positive cells (26.2%) for the cotransfection of 203 and 104. The Cas9 GFP guide RNA5 had 5.21% GFP positive cells under the gated living cells.

Addition of CoCl2: Hypoxia - SEAP assay in HEK cells

1:10 dilution of a 2M CoCl2 stock with PBS. CoCl2 was added to HRE-SEAP approaches in order to induce the promoter, as well as to CMV-SEAP approaches

Approaches: For each approach triplicates were done.

CoCL2 concentration[µM]	volume[µL] of 200mM CoCl2
0	0
100	0.55
150	0.825
200	1.1
250	1.375
300	1.65
500	2.75
1000	5.5

Lentivirus production in HEK cells: Mock, Knock-down plasmid (114,115,116), pIG17_119, pIG17_121

Follow the protocol from Frederike (AG Schamel) PEI Transfection of HEK cells for virus production.

Prepare packaging and PEI mix separately and incubate for 10 min
Mix packaging mix with transferplasmid
Add PEI mix to DNA mix and incubate for 15 min
Add the PEI+DNA mix to cells

Lentiviral packaging cells	Hek293T cells
Plate size	10 cm
Total DNA/plate	12.87 µg
Transfer: gag/pol: env ratio	2:1:1
Construct	6.43 µg
pCMV∆R8.74	3.22 µg
pMD2G	3.22 µg
N/P ration	15

Plasmid number	component	No. of Approaches	concentration[ng/µL]	volume [µL]
pIG17_082	Mock(p526):EF1-GFP	2	68	189.1
pIG17_114	Knock-down plasmid	2	350; 432	18.4; 15
pIG17_115	Knock-down plasmid	1	-	all two tubes
pIG17_116	Knock-down plasmid	2	492; 356	20; 8.5
pIG17_003	Envelope plasmid pMD2G	7	150;68.7;63.7	30;180;80
pIG17_004	Packaging Plasmid pCMV∆R8.74	7	926.6	24.3

Packaging Mastermix

Component	7x [µL]
pIG17_003	290
pIG17_004	24.3
150mM NaCl	3090.43

Follow our protocol of PEI transfection

DNA:PEI ratio = 1:8
Mix DNA with 1 ml serum free DMEM
Add PEI while vortexing
15 min incubation

Component	Mass/approach[µg]
pIG17_003	3.2
pIG17_004	3.2
pIG17_119	6.4
pIG17_121	6.4

03.08.17

Hypoxia - SEAP assay in HEK cells

- gather 200 µl of cell supernatant
- incubate cell supernatant at 65°C for 30min
- centrifuge cell supernatant for 1 min at 1250g
- add 80 µl supernatant into a 96 well plate
- add 100 µl 2x SEAP buffer
- add 20 µl pNPP and remove bubbles carefully
- measure in the plate reader every 30s for 2h

–> Experiment failed and will be repeated

05.08.17

Lentiviral Transduction

Plate 200000 Jurkat cells/well in 6-well plate
Titer Test of virus
Incubation during weekend

Puromycin killing curve

testing of untransfected JK cells and the scramble JK cells that have been transfected with the KO Kit (for each 5 conditions will be tested)
dilution of the Puro stock (10 mg/ml) 1:100 –> 100 µg/ml
cells will be distributed in a 12 well plate with 1 ml of media

concentration of Puro	volume from 100 µg/ml [µl]
0	0
0.125	1.25
0.2	2
0.25	2.5
0.5	5

06.08.17

PEI for HRE-SEAP and CMV-SEAP; repetition of the SEAP assay

cells will be distributed in a 12 well plate on the next day
one master mix for PEI
1 ml DMEM, 30 µl PEI, 10 µg DNA per each approach
cotransfection of CMV-SEAP (95%) and HRE-SEAP (95%) with CMV-GFP (5%)

construct	concentration [ng/µl]	volume added for 9.5 µg [µl]
HRE-SEAP	248.2	38.3
CMV-SEAP	415.2	22.9

construct	concentration [ng/µl]	volume added for 0.5 µg [µl]
CMV-GFP	303.1	1.6

Per Approach: 9µg DNA, 2 mio JK cells

BioRad Electroporation

Approach	plasmid	Capacitance[µF]	Voltage	Resistance	Time Constance [msec]
1	no Plasmid	950	250	∞	20
2	CMV-GFP	950	250	∞	20
3	CMV-GFP	950	250	∞	20
4	Ca construct	950	250	∞	20
5	Ca construct	950	250	∞	20
6	Ca construct	950	250	∞	20
7	Cas9-GFP-gRNA1	950	250	∞	20
8	Cas9-GFP-gRNA1	950	250	∞	20
9	Cas9-GFP-gRNA4	950	250	∞	20
10	Cas9-GFP-gRNA4	950	250	∞	20
11	Cas9-GFP-gRNA5	950	250	∞	20
12	Cas9-GFP-gRNA5	950	250	∞	20
13	Cas9-GFP-scramble	950	250	∞	20
14	Cas9-GFP-scramble	950	250	∞	20

07.08.17

PEI for HRE-SEAP and CMV-SEAP; repetition of the SEAP assay

GFP was observed under the microscope
the cells were evenly distributed onto 12 well plates
induction with CoCl2 after the cells recovered from the transfer
8 conditions per approach, everything done in triplicates

concentration of CoCl2 [µM]	volume added [µl]
0	0
100	0,5
150	0,75
200	1
250	1,25
300	1,5
500	2,5
1000	5

08.08.17

Lentivirus production in HEK cells: Mock, pIG17_133, pIG17_119, pIG17_121

pei_lentivirus_production_08.08.17.xlsx

PEI for HRE-SEAP and CMV-SEAP; repetition of the SEAP assay

The SEAP assay was negative. No changes in the OD for either CMV-SEAP or HRE-SEAP could be observed. Due to these troubles the next test will be postponed till all constructs have undergone another test digestion and sequencing.

10.08.17

Lentiviral transduction

Virus: pIG17_082, pIG17_119, pIG17_121 and pIG17_133
Plate 200000 virus for infection
As control: HEK with 1:1 infection

Cell Sorting: KO-plasmid transfected cells

Cas9-GFP-scramble

Cas9-GFP-gRNA1

Cas9-GFP-gRNA4

Cas9-GFP-gRNA5

11.08.17

BioRad Electroporation

9µg DNA and 2 mio. cells/ approach

Approach	plasmid	Capacitance[µF]	Voltage	Resistance	Time Constance [msec]
1	no Plasmid	950	250	∞	20
2	KO Kit 203+101	950	250	∞	20
3	KO Kit 203+104	950	250	∞	20
4	Lenti-Cas9-Puro	950	250	∞	20

Puromycin killing curve

Day 6 after addition of puromycin: due to low cell numbers, the cells were spun down, resuspended in 100 µl and then counted.

Results for untransfected Jurkat cells and Jurkat cells transfected with the scramble:

Puromycin addition to the electroplated KO Kit cells

Approaches:

1x scramble no treatment
1x scramble 0.2 µg/ml
2x KO Kit 203+101 0.2 µg
2x KO Kit 203+104 0.2 µg

Transfer to a 6 well plate

12.08.17

Puromycin: electroplated KO Kit cells

Due to low cell counts the duplicates were pooled together and the cells transferred to a 12 well plate.

14.08.17

PEI: HRE-SEAP, pWW56

in 10cm plates:

10 µg DNA
1ml DMEM
30 µl PEI

approaches:

HRE-SEAP
pWW56 as a positive SEAP control
untransfected control

construct	concentration [ng/µl]	volume used for 10 µg DNA
HRE-SEAP	420,5	23,78
pWW56	555,5	18,00

15.08.17

SEAP

the cells were evenly distributed onto 12 well plates
induction with CoCl2 after the cells recovered from the transfer
8 conditions per approach, everything done in triplicates

concentration of CoCl2 [µM]	volume added [µl]
0	0
100	0,5
150	0,75
200	1
250	1,25
300	1,5
500	2,5
1000	5

Electroporated KO Kit cells

Due to low cell counts the samples were transferred to a conical 96-well plate

BioRad Electroporation

Approach	plasmid	Capacitance[µF]	Voltage	Resistance	Time Constance [msec]
1	no Plasmid	950	250	∞	20
2	CMV-GFP + CMV-mCherry	950	250	∞	20
3	pIG17_086	950	250	∞	20
4	pIG17_086	950	250	∞	20

Lentivirus Production in HEK cells

Approached by Frederike (AG Schamel)
2 Approaches (pIG17_119 & pIG17_121) with our HEK cells, envelope & packaging plasmid,PEI
1 Approach (pIG17_119) with material from AG Schamel: HEK, envelope & packaging plasmid, PEI

16.08.17

SEAP

The SEAP assay was performed as mentioned before, however, there was a 1:5 dilution included of the HRE-SEAP samples.

For the analysis of the SEAP assay, the averages of the triplicates were calculated and the respective wild-type control subtracted from the HRE-SEAP samples.

For the undiluted HRE-SEAP construct the following graph was obtained:

For the 1:5 dilution the following graph was obtained:

For the 1:5 dilution absorbance values are missing at the start because they were negative and therefore regarded as 0.

The positive control did not work due to the promoter of this construct which was dependent on an input that wasn't given. Furthermore, the samples did not show what we expected, considering that the uninduced control showed a steeper slope than most of the samples. Due to this reason the experiment was repeated.

17.08.17

Lentiviral Transduction with virus-medium

Approaches for Titer test: both normal transduction and spin-infection.
On 8/20: After washing the cells, there was no signal from reporter gene expression.

23.08.17

PEI transfection of HEK cells for lentivirus production

Transferplasmid: pIG17_130 (duplicate)

transfection_pei_lentivirus_23.08.17.xlsx

25.08.17

Concentrate virus (pIG17_130)

Add 8 ml 20% Sucrose (in PBS + 1Mm EDTA) in 50 ml Falcon Tube
Add virus media slowly over sucrose (Use 25ml pippet)
Centrifuge over night at 4000 rpm, 4°C

26.08.17

Lentiviral Transduction (pIG17_130)

Plate 200000 cells/well (HEK & Jurkat) into 6 well plate
Titer of virus: 0, 15 µl, 30 µl

As shown in the picture the transduction of HEK cells showed high efficiency. The cells were also mycoplasma-negative, so that the cells can be sorted at Bioss.

The efficiency of transduction was quite low. The positive signal showed above may come from dead cells. New transduction was done on 8/30 with higher virus titer (45µl and 60µl).

Lentiviral transduction of Jurkat cells with higher virus titer showed higher efficiency than previous try. These cells are sent for microplasma-test and for cell sorting.

30.08.17

PEI transfection of HEK cells for lentivirus production (133&134)

pei_transfection_of_hek_virus_30.08.17.xlsx

02.09.17

PEI transfection of HEK cells for lentivirus production (132)

pei_transfection_of_hek_virus_2.09.17.xlsx

labbooks

04.09.17

BioRad Electroporation

9µg for each approach

Setting: 12 well plate, 1 well set, 1 pulse

Number of approaches	Plasmid	Concentration [ng/µl]	Volume for 9ng [µl]	Capacitance[µF]	Voltage	Resistance	Time Constance [msec]
1	no DNA	0	0	950	250	∞	20
1	CMV GFP, CMV mCherry	327.1, 1200	1376, 3.75	950	250	∞	20
2	pIG17_031	746.9	12.05	950	250	∞	20
2	pIG17_034	1210.9	7.43	950	250	∞	20
2	pIG17_037	851.9	10.56	950	250	∞	20

05.09.17

Washing of the electroporated cells

06.09.17

Sorting of the electroporated cells Gating after:

living cells
single cells
mCherry positive cells

Sorting strategy:
Control igem_gfp_mcherry_jurkat_elektro_tube_001_06092017102918.pdf
pIG17_031 igem_gfp_mcherry_jurkat_31_tube_001_06092017103019.pdf
pIG17_034 igem_gfp_mcherry_jurkat_34_tube_001_06092017110102.pdf
pIG17_037 igem_gfp_mcherry_jurkat_37_tube_001_06092017112901.pdf

Sorting reports:
130 Jurkat: sort_report_06092017122940.pdf
pIG17_034 sort_report_06092017112206.pdf
pIG17_037 sort_report_06092017120252.pdf

07.09.17

Induction of the promoters

Set-up for pIG17_031 - hypoxia

- in a 96 well plate with 200 µl volume:

used concentration of CoCl2: 20 mM
approximately 15000 cells per approach

CoCl2 concentration [µM]	Number of approaches	Volume added [µl]
0	1	0
50	2	0.5
100	2	1
200	2	2
400	2	4
800	2	8

.

Set-up for pIG17_034 - pH

- in a 96 well plate with 200 µl volume:

used concentration of NaOH: 1 M
used concentration of lactate: 0.6 M
used concentration of Forskolin: 10 mM
approximately 15000 cells per approach

Lactate (L), NaOH (N) or Forskolin (F) concentration or pH	Number of approaches	Volume added [µl]
100 µM (F)	1	2
8.17 pH (N)	2	0.4
7.85 pH	2	0
6.54 pH (L)	2	8
6.19 pH (L)	2	10.67

6 well plate with 3ml RPMI and the same concentration of lactate or NaOH as in the 96 well plate but without cells
used in order to measure the pH
amount of cells will be neglected as it is rather low

Set-up for pIG17_037 - VEGF

- in a 96 well plate with 200 µl volume:

used concentration of VEGF: 1 µg/ml
approximately 15000 cells per approach

VEGF concentration [ng/ml]	Number of approaches	Volume added [µl]
0	1	0
2.5	2	0.5
5	2	1
10	2	2
20	2	4
40	2	8

27.08.17 - 07.09.17

Neomycin killing curve of Jurkat cells

11.09.17

PEI transfection of HEK cells for lenvirus production

transfection_pei_11.9.17.xlsx

Viafect - new testing

Jurkat cells with CMV-GFP; transfection mix:

12 well plate
2 mio cells per well in 500 µl medium
0.5 µg DNA per well
Viafect/DNA = 2:1 and 3:1
50 µl total volume per approach (filled up with serum free DMEM)

removal of the medium from pelleted cells
resuspending of pelleted cells in the transfection mix
addition of 500 µl RPMI 1649 with FCS after
1. 0 seconds
2. 4 seconds
3. 8 seconds
4. 16 seconds
5. 32 seconds
transfer of the cell suspension to a 24 well plate

Analysis in the fluorescence microscope showed that this method did not work again

15.09.17

PEI transfection of HEK cells for lenvirus production

Constructs used: pIG17_130, CRE 4x
transfection_pei.pdf

20.09.17

PEI transfection of HEK cells for lenvirus production

Constructs used: NFAT 4x, HRE 4x
transfection_pei_20.9..pdf The concentrated viruses were stored in -80°C freezer as backup.

21.09.17

PEI transfection of HEK cells for SEAP assay

The set up for the SEAP assay was the same as before, however there wild-type controls with the same amount of CoCl2 included.
All samples were done in triplicates

22.09.17

SEAP assay

Induction with CoCl2, the used concentrations of CoCl2 were: 0, 20, 40, 80, 160, 320 and 640 µM.

23.09.17

SEAP assay

The SEAP assay was performed as before, this time we included a cell count of each pooled triplicate.
For the analysis first the averages of the triplicates were calculated and then divided by the cell count. Afterwards, the corresponding wild-type control was subtracted from the samples and all negative values were set to 0.

We could see that only 20, 40 and 80 µM CoCl2 showed a steeper slope in our samples than the 0 µM control.

24.09.17

PEI transfection of HEK cells for lenvirus production

Constructs used: pIG17_134, CRE 4x
transfection_pei_lentivirus_24.9.17.xlsx

24.09.17

HRE testing with stable HEK cell lines

04.10.17

PEI transfection of HEK cells for lenvirus production

Constructs used: pIG17_133, Suiside plasmid transfection_pei_4.10.17.xlsx

09.10.17

CFP test

Due to the problems with our GFP readout we performed a search in BLAST and found out that our eGFP was wrongly annotate and is a eCFP. We also could see that in the FACS. For this we tested our Jurkat knockdown 130 cells, that stably express CFP then. On the left these cells are measured in the CFP channel and on the right in the GFP channel. The depicted cells are gated for living and single cells:

There is a clear shift and our cells are really CFP positive and not GFP positive

CRE Test in Jurkat cells

With Forskolin and IBMX, the cAMP pathway in the cell is activated.

Cell lines: WT Jurkat, stable CRE4X Jurkat
Conditions: untreated, induction with 100 µM Forskolin, induction with 100 µM IBMX, induction with 100 µM Forskolin and 100 µM IBMX.

FACS:
Cells were gated for living, single, mCherry positive cells and then the amount of CFP positive cells was measured:

''

10.10.17

HRE Test in Jurkat cells

HRE Test in HEK cells

12.10.17

CRE Test in HEK cells

On 11th of Oct.: PEI transfection with CMV-TDAG8 plasmid.

CRE Test in Jurkat cells

We obtained the following results after gating for living, single, mCherry positive cells:

VEGF Test in HEK cells

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 CFP-stability at different pH-values

used cells: Jurkat with CMV_CFP
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

CFP 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-CFP (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_CFP (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)-CFP
pIG17_023	Ctla4(330bp)-CFP
pIG17_037	Ctla4(330bp)-CFP-CMV-mCherry
pIG17_086	Ctla4(380bp)-CFP-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	CFP
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

19.07.-27.07.17

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-CFP-CMV-mCherry
- positive control: cotransfected with CMV-CFP and CMV mCherry
- add VEGF
- incubation for 24h at 37°C

17.08.17

PEI transfection of HEK cells

- CMV-CFP from iGEM
- CMV-CFP from tool box

Flow cytometry

- Ctla4-CFP-CMV-mCherry

18.08.17

BioRad electroporation

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

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-CFP 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-CFP-CMV-mCherry
pIG17_008 + pIG17_009	CMV-CFP + CMV-mCherry
pIG17_034 + β2-receptor	pCRE-CFP-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-CFP-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-CFP-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-CFP-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-CFP-CMV-mCherry + L3
pIG17_034 + TDAG8	pCRE-CFP-CMV-mCherry + TDAG8
pIG17_008 + pIG17_009	CMV-CFP + 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-CFP + CMV-mCherry	-
pIG17_034 + TDAG8	pCRE-CFP-CMV-mCherry + TDAG8	+
no DNA	-	-
no DNA	-	+
pIG17_034	pCRE-CFP-CMV-mCherry	-
pIG17_034	pCRE-CFP-CMV-mCherry	+

- after 10 hours: pH change to 6.5, 7.0, 7.5
- plate reader after 1h, 3h, 6h, 12h, 24h
- FACS analysis after 24h

17.09.17

PEI transfection of plasmid pIG17_031

used cells: HEK cells

Plasmid number	component
pIG17_031	HRE-CFP-CMV-mCherry
pIG17_008 + pIG17_009	CMV-CFP + 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)-CFP-CMV-mCherry + TDAG8
	HRE(4x)-CFP-CMV-mCherry
pIG17_008 + pIF17_009	CMV-CFP + 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-CFP-CMV-mCherry, add acid
- cells transiently transfected with HRE-CFP-CMV-mCherry, add CoCl2
- plate reader: no measurable induction of CFP

23.09.17

FACS analysis of HEK cells

- FACS analysis after CRE induction and HRE induction

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)-CFP-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)-CFP-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-CFP-CMV-mCherry, induction with acid (pH 6.7, 7.4, 8.1)
- FACS measurement failed

FACS analysis of CoCl2 treated HEK cells

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

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

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

conditions:

pH	Forskolin + IBMX	other
6.5	-	-
7.1	-	-
7.7	-	-
no acid	Forskolin (100 µM) + IBMX (100 µM)	-
no acid	-	untransfected
no acid	-	without TDAG8

Ctla4(4x)-CFP-CMV-mCherry (stable), CMV-VEGFR2 (transient):

conditions:

VEGF (ng/ml)	Ionomycin (µmol/l)
0	-
25	-
50	-
100	-
-	1.25
-	2.5
-	5.0
-	10.0

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

conditions:

CoCl2 (µmol/ml)
50
100
200
400
600

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

19.10.17

HRE, Ctla4 and HRE induction

- medium change for Ctla4 and HRE cells:
- medium change for CRE:
- fluorescence measurement after 0h, 3h, 6h

labbooks

Lab Notebook Cloning

19.4.17

Miniprep of pIG17_003/4

Miniprep was performed with QIAprep Spin Miniprep Kit.

Test Digestion of pIG17_003/4

Reaction conditions

pIG17_003 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	14
EcoRI-HF	20U/µl	1
CS-Buffer	10x	2
DNA	264 ng/µl	3

pIG17_004 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	15
EcoRI-HF	20U/µl	0.5
XbaI	20U/µl	0.5
CS-Buffer	10x	2
DNA	334 ng/µl	2

Electrophoresis failed and was repeated.

20.4.17

Miniprep of pIG17_001/2

Miniprep was performed with QIAprep Spin Miniprep Kit.

21.4.17

Test Digestion of pIG17_001/2/3/4

Reaction conditions

pIG17_001 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	14
EcoRI-HF	20U/µl	1
CS-Buffer	10x	2
DNA	178 ng/µl	3

pIG17_002 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	15
EcoRI-HF	20U/µl	0.5
XbaI	20U/µl	0.5
CS-Buffer	10x	2
DNA	246 ng/µl	2

pIG17_003 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	14
EcoRI-HF	20U/µl	1
CS-Buffer	10x	2
DNA	156ng/µl	3

pIG17_004 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	14
EcoRI-HF	20U/µl	1
CS-Buffer	10x	2
DNA	155ng/µl	3

22.4.17

Miniprep of pIG17_001/2

Miniprep was performed with QIAprep Spin Miniprep Kit.

Sequencing of pIG17_001/2

sequencing returned successful

23.4.17

Miniprep of pIG17_001/2

Miniprep was performed with QIAprep Spin Miniprep Kit.
Plasmids were used for PEI transfection.

25.4.17

Miniprep of pIG17_008/9

Miniprep was performed with QIAprep Spin Miniprep Kit.

DNA concentrations:
pIG17_008: 1043 ng/µl and 167,4 ng/µl
pIG17_009: 1295,1 ng/µl and 504,3 ng/µl

6.5.17

Test Digestion of pIG17_008/9

Reaction conditions

pIG17_008 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	17
ClaI	20U/µl	0,25
XbaI	20U/µl	0,25
CS-Buffer	10x	2
DNA	896ng/µl	0,5

pIG17_009 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	17
PvuII	20U/µl	0,5
CS-Buffer	10x	2
DNA	765ng/µl	0,5

PCR of pIG17_009

pIG17_009 Total reaction volume 25µl

component	concentration	volume [µL]
H20	-	15,75
oIG17_009 (Primer fw)	10µM	1,25
oIG17_010 (Primer rv)	10µM	1,25
5x Q5 Reaction Buffer	-	5
dNTPs	10mM	0,5
Polymerase Q5 HF	-	0,25
DNA	10ng/µl	1

7.5.17

Test Digestion of pIG17_008

Reaction conditions

pIG17_008 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	17
CalI	20U/µl	0,25
KpnI	20U/µl	0,25
CS-Buffer	10X	2
DNA	798ng/µl	0,5

pIG17_008 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	17
PstI	20U/µl	0,5
CS-Buffer	10x	2
DNA	886ng/µl	0,5

pIG17_008 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	17
XbaI	20U/µl	0,25
NdeI	20U/µl	0,25
CS-Buffer	-	2
DNA	µl	0,5

Gel Extraction of pIG17_009

Gel extraction was performed with QIAquick Gel Extraction Kit.

Concentration of pIG17_009: 51,5 ng/µl

9.5.17

Digestion of pIG17_002

Reaction conditions

pIG17_002 Total reaction volume 50µl

component	concentration	volume [µL]
H20	-	15
NdeI	20U/µl	1,5
AatII	20U/µl	1,5
CS-Buffer	10x	5
DNA	2µg	27

13.5.17

Gel Extraction of pIG17_002 --> SEAP and pA

Gel extraction was performed with QIAquick Gel Extraction Kit.

Concentration of pIG17_002: 84,5 ng/µl

Digestion of pIG17_001/002

Reaction conditions

pIG17_001 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	13
NdeI	20U/µl	0,5
AatII	20U/µl	0,5
10x Fast Digest Green Buffer	-	2
DNA	500ng	4

undigested:

component	concentration	volume [µL]
H20	-	14
10x Fast Digest Green Buffer	-	2
DNA	500ng	4

pIG17_002 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	13
NdeI	20U/µl	0,5
AatII	20U/µl	0,5
10x Fast Digest Green Buffer	-	2
DNA	500ng	4

undigested:

component	concentration	volume [µL]
H20	-	14
10x Fast Digest Green Buffer	-	2
DNA	500ng	4

PCR of pA/SEAP

pIG17_002 Total reaction volume 25µl

component	concentration	volume [µL]
H20	-	15,75
oIG17_012 (Primer fw)	10µM	1,25
oIG17_013 (Primer rv)	10µM	1,25
5x Q5 Reaction Buffer	-	5
dNTPs	10mM	0,5
Polymerase Q5 HF	-	0,25
DNA	10ng/µl	1

pIG17_002 Total reaction volume 25µl

component	concentration	volume [µL]
H20	-	15,75
oIG17_014 (Primer fw)	10µM	1,25
oIG17_015 (Primer rv)	10µM	1,25
5x Q5 Reaction Buffer	-	5
dNTPs	10mM	0,5
Polymerase Q5 HF	-	0,25
DNA	10ng/µl	1

Gel Ex of pA/SEAP

Gel extraction was performed with QIAquick Gel Extraction Kit.
Concentration of SEAP: 45,3 ng/µl
Concentration of pA: 49,1 ng/µl

15.5.17

Digestion of pIG17_002

Reaction conditions

pIG17_002 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	10
NdeI	20U/µl	1
AatII	20U/µl	1
CS-Buffer	10x	2
DNA	105,3 ng/µl	6

undigested: Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	15,5
CS-Buffer	-	2
DNA	-	2,5

Because of the troubles to digest, the digestion was done for 2.5h by 37°C.

PCR of pIG17_009

pIG17_009 Total rection volume 25µl

component	concentration	volume [µL]
H20	-	15,75
oIG17_009 (Primer fw)	10µM	1,25
oIG17_010 (Primer rv)	10µM	1,25
5x Q5 Reaction Buffer	-	5
dNTPs	10mM	0,5
Polymerase Q5 HF	-	0,25
DNA	10ng/µl	1

18.5.17

Gibson Assembly of CMV, mCherry, pA and pSB1C3

pIG17_058 Total rection volume 10µl

component	concentration	volume [µL]
2x Gibson MM	-	5
pSB1C3 Backbone	0,05pmol	2
CMV	0,05pmol	1
mCherry	0,05pmol	1
pA	0,05pmol	1

Placed at 50°C for 1h

Gibson Assembly of pIG17_012, pIG17_013, pIG17_015, pIG17_016

pIG17_012 Total rection volume 10µl

component	concentration	volume [µL]
2x Gibson MM	-	5
pcDNA3.1	0,05pmol	2
HRE	0,05pmol	1
SEAP	0,05pmol	1
pA	0,05pmol	1

Placed at 50°C for 1h

pIG17_013 Total rection volume 10µl

component	concentration	volume [µL]
2x Gibson MM	-	5
pcDNA3.1	0,05pmol	2
HRE	0,05pmol	1
CFP	0,05pmol	1
pA	0,05pmol	1

Placed at 50°C for 1h

pIG17_015 Total rection volume 10µl

component	concentration	volume [µL]
2x Gibson MM	-	5
pcDNA3.1	0,05pmol	2
CRE	0,05pmol	1
SEAP	0,05pmol	1
pA	0,05pmol	1

Placed at 50°C for 1h

pIG17_016 Total rection volume 10µl

component	concentration	volume [µL]
2x Gibson MM	-	5
pcDNA3.1	0,05pmol	2
CRE	0,05pmol	1
eCFP	0,05pmol	1
pA	0,05pmol	1

Placed at 50°C for 1h

PCR of pIG17_027/028/029

PCR of extracted genomic DNA of Jurkat cells to amplify CTLA4

pIG17_027/028/029 Total rection volume 25µl

component	concentration	volume [µL]
H20	-	8
oIG17_048/049/050 (Primer fw) (1000bp/380bp/330bp)	10µM	1,25
oIG17_051 (Primer rv)	10µM	1,25
5x Q5 MM	-	12,5
DNA	10ng/µl	2

Annealing temperatures:
CTLA4 1000bp: 66°C
CTLA4 380bp: 64°C
CTLA4 330bp: 65°C

Gel picture

CTLA4 380bp and 330bp have the expected length. For CTLA4 1000bp we couldn't see any band on the gel.

Gel Extraction of pIG17_028/029

CTLA4 1000bp was not extracted because it couldn't be seen on the gel.
Gel extraction was performed with QIAquick Gel Extraction Kit.

19.5.17

Gibson Assembly of CMV, mCherry delta PstI, pA and pSB1C3

pIG17_058 Total rection volume 10µl

component	concentration	volume [µL]
2x Gibson MM	-	5
pSB1C3 Backbone	0,05pmol	2
CMV	0,05pmol	1
mCherry delta PstI	0,05pmol	1
pA	0,05pmol	1

Placed at 50°C for 1h

21.5.17

Subcloning of CTLA4_330/380

330 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	2
T4 Ligase	-	2
T4 Ligase Reaction buffer	2x	10
pJET2.1	50ng/µl	1
DNA	5ng/µl	5

380 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	2
T4 Ligase	-	2
T4 Ligase Reaction buffer	2x	10
pJET2.1	50ng/µl	1
DNA	5ng/µl	5

Sequencing of pIG17_012/13/15/16

Sequencing returned positive

22.5.17

Test digestion of mCherry and the Gibson Assembly G1.1

Digestion of the constructs was performed to test the function of the enzymes.

Reaction conditions

mCherry Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	13,75
KpnI	20U/µl	0,25
CS-Buffer	10x	2
DNA	-	4

undigested: Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	14
CS-Buffer	-	2
DNA	-	4

undigested without CS-Buffer: Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	16
DNA	-	4

Gibson Assembly G1.1 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	12,25
HindIII	20U/µl	0,25
AflII	20U/µl	0,25
ClaI	20U/µl	0,25
CS-Buffer	10x	2
DNA	-	5

undigested: Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	13
CS-Buffer	-	2
DNA	-	5

Gel picture

Because of the lack of an AafII restriction site in the G1.1 construct there are only two bands to be seen.
All bands have the expected length and the enzymes still work.

23.5.17

Test digestion of CTLA4 380bp and 330bp

Reaction conditions

CTLA4 330 (3,4) and CTLA4 380 (1) Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	12,5
NotI	20U/µl	0,25
NcoI-HF	20U/µl	0,25
CS-Buffer	-	2
DNA	500ng	5

CTLA4 330 (1,2) and CTLA4 380 (3,4) Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	13,5
NotI	20U/µl	0,25
NcoI-HF	20U/µl	0,25
CS-Buffer	-	2
DNA	500ng	4

CTLA4 380 (2) Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	14,5
NotI	20U/µl	0,25
NcoI-HF	20U/µl	0,25
CS-Buffer	-	2
DNA	500ng	3

Undigested samples were done, respectively.

Gel picture

CTLA4 330bp number 1 and also number 1 of CTLA4 380bp were chosen for further procedures.

30.5.17

Obtaining pSAM200 and pMF111 form AG Weber

31.5.17 Test digestion of pSAM200/pMF111

pSAM200 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	13,5
XbaI	20U/µl	0,25
XhoI	20U/µl	0,25
CS-Buffer	10x	2
DNA	170,3ng/µl	4

pMF200 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	13,5
XbaI	20U/µl	0,25
XhoI	20U/µl	0,25
CS-Buffer	10x	2
DNA	133,7ng/µl	4

3.6.17

Midiprep of pIG17_003/082

Midiprep was performed with the Promega PureYield Plasmid Midiprep System Kit.

Miniprep of CTLA4 (330bp, 380bp)

Miniprep was performed with QIAprep Spin Miniprep Kit.

Extension PCR of CTLA4 (330bp, 380bp)

CTLA4 (330bp)
Total rection volume 25µl

component	concentration	volume [µL]
H20	-	9
oIG17_105 (Primer fw)	10µM	1,25
oIG17_106 (Primer rv)	10µM	1,25
5x Q5 MM	-	12,5
DNA	10ng/µl	1

CTLA4 (380bp)
Total rection volume 25µl

component	concentration	volume [µL]
H20	-	9
oIG17_107 (Primer fw)	10µM	1,25
oIG17_106 (Primer rv)	10µM	1,25
5x Q5 MM	-	12,5
DNA	10ng/µl	1

Annealing temperatures:
CTLA4 380bp: 59°C
CTLA4 330bp: 65°C

4.6.17

Extension PCR of CTLA4 (330bp, 380bp)

Because the extension PCR didn't work the day before, we performed another one.

CTLA4 (330bp)
Total rection volume 25µl

component	concentration	volume [µL]
H20	-	9
oIG17_105 (Primer fw)	10µM	1,25
oIG17_106 (Primer rv)	10µM	1,25
5x Q5 MM	-	12,5
DNA	10ng/µl	1

CTLA4 (380bp)
Total rection volume 25µl

component	concentration	volume [µL]
H20	-	9
oIG17_107 (Primer fw)	10µM	1,25
oIG17_106 (Primer rv)	10µM	1,25
5x Q5 MM	-	12,5
DNA	10ng/µl	1

Annealing temperatures:
CTLA4 380bp: 59°C
CTLA4 330bp: 65°C

Gel Extraction of CTLA4 (330bp, 380bp) of the extension PCR

Gel extraction was performed with QIAquick Gel Extraction Kit.

Gibson Assembly of CTLA4, GFP/SEAP, pA and BB - pIG17_019/020/022/023

CTLA4 (330bp) and GFP - pIG17_023
Total rection volume 10µl

component	concentration	volume [µL]
2x Gibson MM	-	5
pSB1C3 Backbone	0,06pmol	0,75
CTLA4 (330bp)	0,06pmol	1
GFP	0,06pmol	0,5
pA	0,06pmol	1
H2O	-	1,75

CTLA4 (330bp) and SEAP - pIG17_020
Total rection volume 10µl

component	concentration	volume [µL]
2x Gibson MM	-	5
pSB1C3 Backbone	0,06pmol	0,75
CTLA4 (330bp)	0,06pmol	1
SEAP	0,06pmol	0,75
pA	0,06pmol	1
H2O	-	1,5

CTLA4 (380bp) and GFP - pIG17_022
Total rection volume 10µl

component	concentration	volume [µL]
2x Gibson MM	-	5
pSB1C3 Backbone	0,06pmol	0,75
CTLA4 (380bp)	0,06pmol	0,5
GFP	0,06pmol	0,5
pA	0,06pmol	1
H2O	-	2,25

CTLA4 (380bp) and SEAP - pIG17_019
Total rection volume 10µl

component	concentration	volume [µL]
2x Gibson MM	-	5
pSB1C3 Backbone	0,06pmol	0,75
CTLA4 (380bp)	0,06pmol	0,5
SEAP	0,06pmol	0,75
pA	0,06pmol	1
H2O	-	2

Placed at 50°C for 1h

Transformation pIG17_019/020/022/023/030

Transformation was done with DH5alpha Z-competent cells for all CTLA4-GFP/SEAP Gibson assemblies.
pIG17_030 was done again because there were no colonies on the dish.

5.6.17

Midiprep of pIG17_004/008/009

Midiprep was performed with the Promega PureYield Plasmid Midiprep System Kit.

Gel

Because of the trouble to get a transformation of pIG17_030, the Gibson assembly and the used constructs were loaded on a gel, in order to find out where the problem lies.

Result of Gibson assembly

CTLA4 330 with GFP did growth
CTLA4 380 with GFP did growth
CTLA4 330 with SEAP did not growth
CTLA4 380 with SEAP did not growth

6.6.17

Extention PCR of LUC

Extention PCR made to get rid of unwanted the cutting site

Miniprep of CTLA4_GFP (pIG17.022, pIG17.023)

Miniprep with JenaBioscience. The yield was too poor to work with. New cultures are needed. o/n are done.

Sequencing of SEAP

SEAP construct (19, 20) are send to sequencing.

Sequencing analysis of pCRE and pHRE (pIG17.006, pIG17.007)

The results:

Pick of CTLA4 330_SEAP (pIG17.020)

Test digest of CTLA4 330 and 380 SEAP & GFP (pIG17.019, pIG17.020, pIG17.022, pIG17.023)

Construct		Enzym	Size	Size undigested
CTLA4 330	GFP	HindI II	2210bp, 925bp	3127pb
	SEAP	HindI II	2210pb, 1726pb	3928pb
CTLA4 380	GFP	HindI II	2260pb, 929pb	3177pb
	SEAP	HindI II	2260pb, 1726pb	3978pb

Transformation of HRE_SEAP (pIG17.030)

Transformation made with 5μl. Did not worked.

Test digest of Luceferase

Concentration: 462ng/μl

	Volumina for digest	Volumina for negative control
DNA	1,1μl	1,1μl
Buffer	2μl	2μl
Water	16,4μl	16,9μl
Enzyme 1	0,25μl
Enzyme 2	0,25μl
Total	20μl	20μl

Used enzyme: 1:XbaI: 37°C, 2:NotI:37°C Buffer: NEBuffer 3.1 DNA used: 500ng

Test digest of pIG17.019, pIG17.020, pIG17.022, pIG17.023

	330 GFP	330 SEAP	380 GFP	330 SEAP	380 SEAP
DNA	250ng	250ng	250ng	250ng	250ng
Water
Enzyme	0,5μl	0,5μl	0,5μl	0,5μl	0,5μl
Buffer	2μl	2μl	2μl	2μl	2μl
Total	20μl	20μl	20μl	20μl	20μl

On the gel: Loading dye: 5μl

Midi Prep of CMV (pIG17.008, pIG17.009)

Midiprep done with Promega PureYield™ Plasmid Midiprep System kit

	1.elution	2.elution
pIG17.008	142,1ng/μl	50,1ng/μl
pIG17.009	89,3ng/μl	64,1ng/μl

7.6.17

Miniprep of pIG17_019/020/022/023

Miniprep was performed with Jena Bioscience Kit.

Results

Transformation of HRE SEAP with mCherry didn't grow

Test digestof pIG17_019/020/022/023

Test diget with HindIII
DNA concentration of 500ng/μl (4μl of DNA) 10xPuffer (2μl) H2O till 20μl in the PCR tube.

GATC sequencing

3x HIF1-a with 30ng/μl with the primer oIG17_119 (3μl with 26μl H2O)

8.6.17

Trouble shooting of the Promega Midiprep kit

Extention PCR with mCherry and pIG17_012

9.6.17

Test Digestion of pIG17_019/020/022/023

Reaction conditions

pIG17_019/020: Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	17,5 - x
Nde I	20U/µl	0,25
Cla I	20U/µl	0,25
CS-Buffer	-	2
DNA	500 ng	x

pIG17_022/023: Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	17,5 - x
Nco I	20U/µl	0,25
Cla I	20U/µl	0,25
CS-Buffer	-	2
DNA	500 ng	x

Undigested:
Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	18 - x
CS-Buffer	-	2
DNA	500 ng	x

Incubation for 1h at 37 degrees

Gel extraction of pIG17_012 and mCherry

DNA	Gel	QG	Isopropanol
pIG17_012	0,12g	240μl	81,16
mCherry	0,08g	360μl	122,4μl

Midiprep of pIG17_008 and pIG17_009

2 bands on the gel : Contamination!

14.6.17

Test Digestion of SDM of LUC

SDM of LUC Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	13,75
BspEI	20U/µl	0,25
Tango Buffer	10x	2
DNA	151,7ng/µl	4

Gibson assembly pIG17_31/34/37/86

pIG17_31 Total rection volume 10µl

component	concentration	volume [µL]
2x Gibson MM	-	5
pcDNA3.1	0,05pmol	2
pIG17_012	0,05pmol	1
pIG17_058	0,05pmol	1

pIG17_34 Total rection volume 10µl

component	concentration	volume [µL]
2x Gibson MM	-	5
pcDNA3.1	0,05pmol	2
pIG17_013	0,05pmol	1
pIG17_058	0,05pmol	1

pIG17_37 Total rection volume 10µl

component	concentration	volume [µL]
2x Gibson MM	-	5
pcDNA3.1	0,05pmol	2
pIG17_015	0,05pmol	1
pIG17_058	0,05pmol	1

pIG17_86 Total rection volume 10µl

component	concentration	volume [µL]
2x Gibson MM	-	5
pcDNA3.1	0,05pmol	2
pIG17_016	0,05pmol	1
pIG17_058	0,05pmol	1

12.6.17

Midiprep of pIG17_008/009

Midiprep was performed with the Promega Kit.

Gibson Assembly of Luciferase with SDM

Total rection volume 24µl

component	concentration	volume [µL]
2x Gibson MM	-	12
Luciferase Backbone	0,06pmol	7,5
PCR product 1 of Luciferase	0,06pmol	1
PCR product 2 of Luciferase	0,06pmol	2,5
PCR product 3 of Luciferase	0,05pmol	0,5
Water	-	0,5

Placed at 50°C for 1h

6.7.17

Mini Prep of HIF1 - alpha fusion proteins (pmf111/psb1c3/LUC delta BspEI)

Miniprep done with Promega PureYield™ Plasmid Miniprep System kit

	concentration
pmf111	48,1ng/μl
psb1c3	107,6ng/μl
LUC delta BspEI	289,6ng/μl

Miniprep of pIG17_003/4

Miniprep was not performed due to the fact that the overnight culture got spilled out.

PCR of HIF1 - alpha fusion proteins (psb1c3/LUC delta BspEI)

psb1c3
Total rection volume 25µl

component	concentration	volume [µL]
H20	-	9
oIG17_198 (Primer fw)	10µM	1,25
oIG17_197 (Primer rv)	10µM	1,25
5x Q5 MM	-	12,5
DNA	10ng/µl	1

LUC delta BspEI
Total rection volume 25µl

component	concentration	volume [µL]
H20	-	9
oIG17_101 (Primer fw)	10µM	1,25
oIG17_202 (Primer rv)	10µM	1,25
5x Q5 MM	-	12,5
DNA	10ng/µl	1

LUC delta BspEI
Total rection volume 25µl

component	concentration	volume [µL]
H20	-	9
oIG17_203 (Primer fw)	10µM	1,25
oIG17_100 (Primer rv)	10µM	1,25
5x Q5 MM	-	12,5
DNA	10ng/µl	1

17.6.17

Testdigestion of pIG17_31/34/37/86

pIG17_031 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	13,75
BspEI	20U/µl	0,25
KpnI	20U/µl	0,25
Tango Buffer	10x	2
DNA	120,7ng/µl	4

pIG17_034 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	13,75
BspEI	20U/µl	0,25
KpnI	20U/µl	0,25
Tango Buffer	10x	2
DNA	130,5ng/µl	4

pIG17_037 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	13,75
BspEI	20U/µl	0,25
KpnI	20U/µl	0,25
Tango Buffer	10x	2
DNA	111,7ng/µl	4

pIG17_087 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	13,75
BspEI	20U/µl	0,25
KpnI	20U/µl	0,25
Tango Buffer	10x	2
DNA	101,9ng/µl	4

Positive clones were send to sequencing

18.6.17

Gibson Assembly of pIG17_063/064/065

pIG17_063 Total rection volume 10µl

component	concentration	volume [µL]
2x Gibson MM	-	5
pcDNA3.1	0,05pmol	1
HRE	0,05pmol	1
LUC	0,05pmol	1
pA	0,05pmol	1
pIG17_058	0,05pmol	1

pIG17_064 Total rection volume 10µl

component	concentration	volume [µL]
2x Gibson MM	-	5
pcDNA3.1	0,05pmol	1
CRE	0,05pmol	1
LUC	0,05pmol	1
pA	0,05pmol	1
pIG17_058	0,05pmol	1

pIG17_065 Total rection volume 10µl

component	concentration	volume [µL]
2x Gibson MM	-	5
pcDNA3.1	0,05pmol	1
NFAT b.s.	0,05pmol	1
LUC	0,05pmol	1
pA	0,05pmol	1
pIG17_058	0,05pmol	1

22.06.17

Test digestion of pIG17_063/064/065

pIG17_063 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	13,75
BspEI	20U/µl	0,25
KpnI	20U/µl	0,25
Tango Buffer	10x	2
DNA	100,7ng/µl	4

pIG17_064 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	13,75
BspEI	20U/µl	0,25
KpnI	20U/µl	0,25
Tango Buffer	10x	2
DNA	120,5ng/µl	4

pIG17_064 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	13,75
BspEI	20U/µl	0,25
KpnI	20U/µl	0,25
Tango Buffer	10x	2
DNA	98,1ng/µl	4

Positive clones were sent to sequencing

28.6.17

Digestion of pIG17_31/34/37/86 for ligation into Transferplasmid

pIG17_031 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	13,75
ClaI	20U/µl	0,25
KpnI	20U/µl	0,25
Tango Buffer	10x	2
DNA	120,1ng/µl	4

pIG17_034 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	13,75
ClaI	20U/µl	0,25
KpnI	20U/µl	0,25
Tango Buffer	10x	2
DNA	160,8ng/µl	4

pIG17_037 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	13,75
ClaI	20U/µl	0,25
KpnI	20U/µl	0,25
Tango Buffer	10x	2
DNA	143,3ng/µl	4

pIG17_086 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	13,75
ClaI	20U/µl	0,25
KpnI	20U/µl	0,25
Tango Buffer	10x	2
DNA	198,5ng/µl	4

Transferplasmid Total reaction volume 50µl

component	concentration	volume [µL]
H20	-	30,25
BspEI	20U/µl	0,5
KpnI	20U/µl	0,5
Tango Buffer	10x	2
DNA	300,4ng/µl	4

T4 Ligation of pIG17_31/34/37/87 into Transfer plasmid

pIG17_31 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	2
T4 Ligase	-	2
T4 Ligase Reaction buffer	2x	10
Transfer plasmid	70ng/µl	1
pIG17_031	13ng/µl	5

pIG17_34 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	2
T4 Ligase	-	2
T4 Ligase Reaction buffer	2x	10
Transfer plasmid	70ng/µl	1
pIG17_034	15ng/µl	5

pIG17_37 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	2
T4 Ligase	-	2
T4 Ligase Reaction buffer	2x	10
Transfer plasmid	70ng/µl	1
pIG17_037	13ng/µl	5

pIG17_87 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	2
T4 Ligase	-	2
T4 Ligase Reaction buffer	2x	10
Transfer plasmid	70 ng/µl	1
pIG17_087	5ng/µl	5

2.7.17

Gibson Assembly of HRE-GFP-pA-SV40-Neomycin/Zeozin

HRE-GFP-pA-SV40-Neomycin Total rection volume 10µl

component	concentration	volume [µL]
2x Gibson MM	-	5
pcDNA3.1	0,05pmol	1
pIg17_013	0,05pmol	1
SV40	0,05pmol	1
Neomycin	0,05pmol	1
pA	0,05pmol	1

HRE-GFP-pA-SV40-Zeozinin Total rection volume 10µl

component	concentration	volume [µL]
2x Gibson MM	-	5
pcDNA3.1	0,05pmol	1
pIg17_013	0,05pmol	1
SV40	0,05pmol	1
Zeocin	0,05pmol	1
pA	0,05pmol	1

03.07.17

Gibson assembly of SV40-TetR-pA

HRE-GFP-pA-SV40-Neomycin Total rection volume 10µl

component	concentration	volume [µL]
2x Gibson MM	-	5
pcDNA3.1	0,05pmol	2
SV40	0,05pmol	1
VP16-TetR	0,05pmol	1
pA	0,05pmol	1

7.7.17

Mini Prep of pIG17_031/034/086/119

Miniprep done with Promega PureYield™ Plasmid Miniprep System kit

	concentration
pIG17.031	46,6ng/μl
pIG17.034	45,7ng/μl
pIG17.086	47,9ng/μl
pIG17.119	390,8ng/μl

PCR of HIF1 - alpha fusion proteins (pMF111/pSB1C3(standard part we got from iGEM))

pMF111 #1
Total rection volume 25µl

component	concentration	volume [µL]
H20	-	9
oIG17_177 (Primer fw)	10µM	1,25
oIG17_211 (Primer rv)	10µM	1,25
5x Q5 MM	-	12,5
DNA	10ng/µl	1

pMF111 #3
Total rection volume 25µl

component	concentration	volume [µL]
H20	-	9
oIG17_150 (Primer fw)	10µM	1,25
oIG17_176 (Primer rv)	10µM	1,25
5x Q5 MM	-	12,5
DNA	10ng/µl	1

pSB1C3 #2
Total rection volume 25µl

component	concentration	volume [µL]
H20	-	9
oIG17_198 (Primer fw)	10µM	1,25
oIG17_197 (Primer rv)	10µM	1,25
5x Q5 MM	-	12,5
DNA	10ng/µl	1

Gel picture

Midiprep of 31, 34, 86, 119

Midiprep with Promega Midiprep kit

pIG17_031	6,0ng/μl
pIG17_034	4,4ng/μl
pIG17_086	9,4ng/μl	6,9ng/μl
pIG17_119	156ng/μl	167ng/μl

labbooks

8.7.17

Gibson Assembly of TetO/HRE/CRE/NFAT b.s.-HIF/HIF-LUC-CMV-mCherry

TetO-HIF-LUC-pA-CMV-mCherry Total rection volume 10µl

component	concentration	volume [µL]
2x Gibson MM	-	6
pcDNA3.1	0,05pmol	1
TetO	0,05pmol	1
HIF	0,05pmol	1
LUC	0,05pmol	1
pA	0,05pmol	1
pIG17_058	0,05pmol	1

HRE-HIF-pA-CMV-mCherry Total rection volume 10µl

component	concentration	volume [µL]
2x Gibson MM	-	5
pcDNA3.1	0,05pmol	1
HRE	0,05pmol	1
HIF	0,05pmol	1
pA	0,05pmol	1
pIG17_058	0,05pmol	1

CRE-HIF-pA-CMV-mCherry Total rection volume 10µl

component	concentration	volume [µL]
2x Gibson MM	-	5
pcDNA3.1	0,05pmol	1
CRE	0,05pmol	1
HIF	0,05pmol	1
pA	0,05pmol	1
pIG17_058	0,05pmol	1

NFAT bs 330-HIF-pA-CMV-mCherry Total rection volume 10µl

component	concentration	volume [µL]
2x Gibson MM	-	5
pcDNA3.1	0,05pmol	1
NFAT b.s. 330	0,05pmol	1
HIF	0,05pmol	1
pA	0,05pmol	1
pIG17_058	0,05pmol	1

NFAT bs 380-HIF-pA-CMV-mCherry Total rection volume 10µl

component	concentration	volume [µL]
2x Gibson MM	-	5
pcDNA3.1	0,05pmol	1
NFAT b.s. 380	0,05pmol	1
HIF	0,05pmol	1
pA	0,05pmol	1
pIG17_058	0,05pmol	1

9.7.17

Bacterial Glycerol Stocks for Long-term Storage of Plasmids

pIG17_037
pIG17_086
pIG17_119

Mini Prep of pIG17_118/120/121

Miniprep done with Promega PureYield™ Plasmid Miniprep System kit

	concentration
pIG17_118	51,6ng/μl
pIG17_120 #1	50ng/μl
pIG17_120 #2	56ng/μl
pIG17_121 #1	208,3ng/μl
pIG17_121 #2	56ng/μl

10.7.17

Mini Prep of pIG17_31 zeomycin and neomycin/pIG17_097

Miniprep done with Promega PureYield™ Plasmid Miniprep System kit

	concentration
pIG17_031 zeo	107,9ng/μl
pIG17_031 neo	25,7ng/μl
pIG17_097 1 #1	30,8ng/μl
pIG17_097 1 #2	34,2ng/μl
pIG17_097 2 #1	38,4ng/μl
pIG17_097 2 #2	40,0ng/μl
pIG17_097 2 #3	32,3ng/μl

12.7.17

Mini Prep of TetO-Luc-HIF/TetO-HIF-Luc/pIG17_031 TetR

Miniprep done with Promega PureYield™ Plasmid Miniprep System kit

	concentration
pIG17_031 TetR	22,0ng/μl
TetO-Luc-HIF #1	32,4ng/μl
TetO-HIF-Luc #1	174,5ng/μl

13.7.17

Mini Prep of pCRE-HIF/CTLA4-HIF

Miniprep done with Promega PureYield™ Plasmid Miniprep System kit

	concentration
pCRE-HIF #3	28,3ng/μl
pCRE-HIF #2	30,1ng/μl
pCRE-HIF #1	30,4ng/μl
CTLA4-HIF(380) #1	32,1ng/μl
CTLA4-HIF(380) #2	33,1ng/μl
CTLA4-HIF(380) #3	32,8ng/μl
CTLA4-HIF(330) #2	29,9ng/μl

15.7.17

Ligation of HRE4/CRE4/NFAT bs*4 into pIG17_031

HRE*4 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	6
T4 Ligase	-	2
T4 Ligase Reaction buffer	2x	10
pIG17_031	65ng/µl	1
HRE*4	10ng/µl	1

CRE*4 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	6
T4 Ligase	-	2
T4 Ligase Reaction buffer	2x	10
pIG17_031	65ng/µl	1
CRE*4	10ng/µl	1

NFAT bs*4 Total reaction volume 20µl

component	concentration	volume [µL]
H20	-	6
T4 Ligase	-	2
T4 Ligase Reaction buffer	2x	10
pIG17_031	65ng/µl	1
NFAT bs*4	10ng/µl	1

16.7.17

Gibson Assembly of HRE4/CRE4/NFAT bs*4-CFP-CMV-mCherry

HRE*4-CFP-CMV-mCherry Total rection volume 10µl

component	concentration	volume [µL]
2x Gibson MM	-	5
Transfer vector	0,05pmol	2
HRE*4	0,05pmol	1
CFP	0,05pmol	1
pIG17_058	0,05pmol	1

CRE*4-CFP-CMV-mCherry Total rection volume 10µl

component	concentration	volume [µL]
2x Gibson MM	-	5
Transfer vector	0,05pmol	2
CRE*4	0,05pmol	1
CFP	0,05pmol	1
pIG17_058	0,05pmol	1

NFAT bs*4-CFP-CMV-mCherry Total rection volume 10µl

component	concentration	volume [µL]
2x Gibson MM	-	5
Transfer vector	0,05pmol	2
NFAT bs*4	0,05pmol	1
CFP	0,05pmol	1
pIG17_058	0,05pmol	1

19.7.17

Mini Prep of pCRE-HIF/CTLA4-HIF/pIG17_096

Miniprep done with Promega PureYield™ Plasmid Miniprep System kit

	concentration
pCRE-HIF	26,7ng/μl
pCRE-HIF	187,7ng/μl
pCRE-HIF	153,0ng/μl
pCRE-HIF	102,0ng/μl
CTLA4-HIF(380)	32,4ng/μl
CTLA4-HIF(380)	340,1ng/μl
CTLA4-HIF(380)	273,5ng/μl
CTLA4-HIF(380)	70,4ng/μl
CTLA4-HIF(380)	88,81ng/μl
CTLA4-HIF(380)	59,9ng/μl
CTLA4-HIF(330)	29,0ng/μl
CTLA4-HIF(330)	315,2ng/μl
CTLA4-HIF(330)	98,8ng/μl
CTLA4-HIF(330)	344,6ng/μl
CTLA4-HIF(330)	135,1ng/μl
pIG17_096	342,8ng/μl
pIG17_096	31,3ng/μl
pIG17_096	71,3ng/μl
pIG17_096	99,7ng/μl
pIG17_096	55,8ng/μl

Sequencing of pCRE-HIF/CTLA4-HIF 330/CTLA4-HIF 330/pIG17_096/pIG17_097/pIG17_118/pIG17_120

plasmid number	primer
pCRE-HIF	oIG17_166/018/164/174/175/119
CTLA4-HIF(380)	oIG17_166/018/164/174/175/119
CTLA4-HIF(330)	oIG17_166/018/164/174/175/119
pIG17_096	oIG17_174/175/169/170/018
pIG17_097	oIG17_054
pIG17_118	oIG17_019
pIG17_120	oIG17_019

21.7.17

Mini Prep of HRE x Mult-GFP-CMV-mCherry/CRE x Mult-GFP-CMV-mCherry/HRE ecFP zeo/TetR VP16 neo

Miniprep done with Promega PureYield™ Plasmid Miniprep System kit

	concentration	curve
HRE x Mult-GFP-CMV-mCherry #1	39,4ng/μl
HRE x Mult-GFP-CMV-mCherry #2	28,9ng/μl
HRE x Mult-GFP-CMV-mCherry #3	28,9ng/μl
HRE x Mult-GFP-CMV-mCherry #4	44,6ng/μl
CRE x Mult-GFP-CMV-mCherry #1	18,9ng/μl
CRE x Mult-GFP-CMV-mCherry #2	49,8ng/μl
CRE x Mult-GFP-CMV-mCherry #3	20,9ng/μl
CRE x Mult-GFP-CMV-mCherry #4	25,2ng/μl
HRE ecFP zeo #1	35,2ng/μl
HRE ecFP zeo #2	26,4ng/μl
HRE ecFP zeo #3	36,2ng/μl
HRE ecFP zeo #4	17,3ng/μl
TetR VP16 neo #1	21,7ng/μl
TetR VP16 neo #2	30,4ng/μl
TetR VP16 neo #3	66,7ng/μl
TetR VP16 neo #4	28,1ng/μl

we also went upstairs to check our concentrations with Toolbox Nanodrop but there wasn't much difference
Denis asked Nicole if she can do a Miniprep for us with her kit
Nicole suggested to try to use the buffer instead of water to see if it will change something

24.7.17

Mini Prep of pIG17_012/013/015/016

Miniprep done with Promega PureYield™ Plasmid Miniprep System kit

	concentration	curve
pIG17_012	187,1ng/μl
pIG17_013	53,9ng/μl
pIG17_015	49,1ng/μl
pIG17_016	26,1ng/μl

29.7.17

Mini Prep of pIG17_003/031/119/

Miniprep done with Promega PureYield™ Plasmid Miniprep System kit (in 40μl Wasser eluiert)

	concentration
pIG17_003	16,9ng/μl
pIG17_003	90,8ng/μl
pIG17_003	144,0ng/μl
pIG17_004	154,9ng/μl
pIG17_004	280,7ng/μl
pIG17_031	541,1ng/μl (!)
pIG17_031	257,1ng/μl (!)
pIG17_031	336,1ng/μl (!)
pIG17_034	23,0ng/μl (!)
pIG17_119	318,6ng/μl (!)
pIG17_119	19,3ng/μl
pIG17_119	31,9ng/μl
pIG17_119	12,1ng/μl
pIG17_119	53,2ng/μl
pIG17_119	314,0ng/μl (!)
HRE GFP	109,9ng/μl
HRE GFP	43,9ng/μl
HRE GFP	89,9ng/μl
HRE GFP	147,1ng/μl
HRE GFP	104,3ng/μl
HRE GFP	67,9ng/μl (!)
HRE GFP	125,2ng/μl
HRE GFP	153,1ng/μl
HRE GFP	318,2ng/μl
HRE GFP	294,9ng/μl
HRE GFP	148,6ng/μl
HRE GFP	82,1ng/μl
HRE GFP	423,9ng/μl (!)
HRE GFP	427,2ng/μl (!)

30.7.17

Mini Prep of pIG17_503/Tet-GFP

Miniprep done with Promega PureYield™ Plasmid Miniprep System kit (in 40μl Wasser eluiert)

	concentration	curve
pIG17_503	242,6ng/μl
Tet-GFP	255,1ng/μl

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Difference between revisions of "Team:Freiburg/Notebook"

Revision as of 18:32, 20 October 2017

Lab Notebook Cell Culture

PEI transfection of HEK cells

13.05.17

PEI transfection of HEK, Jurkat, HPB-ALL and HUT78 cells

23.05.17

PEI transfection of HEK, Jurkat, HPB-ALL and HUT78 cells

Lipotransfection of Jurkat, HPB-ALL and HUT78 cells

30.05.17

BioRad electroporation

preparation for lentiviral transduction

06.06.17

Production of Lentivirus

07.06.17

Amaxa electroporation

Production of Lentivirus

BioRad electroporation

08.06.17

Production of Lentivirus

Flow Cytometry

09.06.17

Lentiviral transduction

10.06.17

BioRad electroporation

12.06.17

Lentiviral transduction

Flow Cytometry for BioRad electroporation on June 10th

13.06.17

Flow Cytometry for lentiviral infected cells

BioRad Electroporation

14.06.17

Amaxa electroporation

Flow Cytometry for BioRad electroporated JK cells

15.06.17

Flow Cytometry for BioRad electroporated Hut78 cells and Amaxa electroporated T cells

16.06.17

Mycoplasma test(lentiviral transduced Jurkat and Hut78)

17.06.17

BioRad Electroporation with JK cells

PEI transfection with HEK cells

19.06.17

Hypoxia test (CoCl2)

BioRad Electroporation

20.06.17

BioRad electroporation

21.06.17

Mycoplasma-Test (Stock cultures)

Flow Cytometry (BioRad on Jun.19th)

24.06.17

PEI transfection (pIG17_013) for hypoxia test

25.06.17

Hypoxia test (CoCl2)

BioRad Electroporation

26.06.17

Hypoxia test (CoCl2)

Flow Cytometry (Hypoxia test & BioRad)

Hypoxia test

BioRad Electroporation

27.06.17

BioRad Electroporation

GFP Plasmid: pIG17_009

Knock out

28.06.17

Flow Cytometry: electroporation on June 27th

29.06.17

Production of Lentivirus

30.06.17

Production of Lentivirus

01.07.17

Production of Lentivirus

Lentiviral transduction

PEI transfection with HEK for Hypoxia Test

BioRad Electroporation: Repeat pGFP transfection

02.07.17

CoCl2 treatment with Jurkat cells

Repeat Hypoxia test (24h after PEI)

Flow Cytometry (BioRad on July 1st)

03.07.17

Repeat Hypoxia test (48h after PEI)