labor:cell_culture - iGEM 2017

Lab Notebook Knockout

06.05.2017

Retransformation of lenti-CAS9-puro plasmid

A heatshock transformation of XL10 gold was performed with the lenti-CAS9-puro plasmid.

07.05.2017

Producing overnight cultures

Two colonies were observed.
5 ml LB-Medium containing the appropriate antibiotic was inoculated and incubated overnight at 37°C, 180rpm.

Test digest of lenti-CAS9-puro

0.66 μg DNA ( 3ul ~ 220ng/μl)
0.25 μl NcoI-HF
1 μl CutSmart 10x buffer
5.75μl H20

Digestion at 37°C for 1 h.
→ Gel did not show separation of bands. Predicted fragments: ~6,100bp; 7,400bp

08.05.2017

Miniprep of lenti-CAS9-puro

Plasmid was extracted using the provided protocol (Quiagen Kit).
DNA was eluated in 30μl H2O.

09.05.2017

Test digest of lenti-CAS9-puro

2 μl Plasmid
0.25 μl NcoI-HF
1 μl CutSmart 10x buffer
6.75 μl H20

Digest was performed with lenti-CAS9-puro plasmids obtained from Minipreps and from the origial stock.
Digestion at 37°C for 1h.

Gel: 1.15h at 180V. Expected bands could be observed in all samples.

10.5.2017

Test digestion of Cathomen Cas9 plasmid

11.05.2017

Retransformation of knockdown plasmids

A heatshock transformation of XL10 gold was performed with the following plasmids: SDEN, SBEN, EDEN.
Plasmids were provided by AG Schamel.

12.05.2017

Producing overnight cultures of SDEN, SBEN, EDEN

5 ml LB-Medium containing the appropriate antibiotic was inoculated and incubated overnight at 37°C and 30°C; 180rpm.

13.05.2017

Retransformation of CAS9-GFP plasmid

A heatshock transformation of XL10 gold was performed with the CAS9-GFP plasmid.

Miniprep of SDEN, SBEN, EDEN

Plasmid was extracted using the provided protocol (Quiagen Kit).

Temp.	Prep 1	Prep 2	Prep 3
30°C	17,5 ng/μl	24,0ng/μl	34,0ng/μl
37°C	53,1ng/μl	18,4g/μl	11,0ng/μl

14.05.2017

Producing overnight cultures CAS9-GFP

5 ml LB-Medium containing the appropriate antibiotic was inoculated and incubated overnight at 37°C; 180rpm.

15.05.2017

Miniprep of CAS9-GFP

Plasmid was extracted using the provided protocol (Quiagen Kit).
Prep 1: 404.4 ng/μl
Prep 2: 495.5 ng/μl

Testdigest of CAS9-GFP

	origial stock	Prep 1	Prep 2
DNA	0.25 μl	1 μl	1 μl
NcoI-HF	0.25 μl	0.25 μl	0.25 μl
CutSmart 10x	1 μl	1 μl	1 μl
H2O	7.75 μl	6.75 μl	6.75 μl

Digestion at 37°C for 1h.
Expected fragments: ~500bp; 8,500bp.
Expected fragments were observed in all samples.

18.05.2017

PCR for HIF1A exons

2 μl DNA
1.25 μl Primer Fwd
1.25 μl Primer Rev
12.5 μl KAPA 2x

As template gDNA from JK, HUT and HPB All cells was used.
PCR program:
3 min. 95°C
15 sec. 95°C
15 sec. 54°C
20 sec. 72°C
10 min. 72°C

The expected bands were cut out of the gel and purified the next day; gel was stored at 4°C overnight.

19.05.2017

Gel extraction

Gel extraction was performed following the manufacturer's protocol (Quiagen Gel extraction kit).

Cell line	Exon 5	Exon 6	Exon 8	Exon 10
JK	16.6 ng/μl	33.5 ng/μl	31.5 ng/μl	20.6 ng/μl
HPB	23.1 ng/μl	34.6 ng/μl	26.0 ng/μl	12.2 ng/μl
HUT	14.1 ng/μl	15.9 ng/μl	25.2 ng/μl	18.6 ng/μl

Retransformation of KO-Kit plasmids

A heatshock transformation of XL10 gold was performed with the plasmids 101,104,203.

20.05.2017

Miniprep and Midiprep of several plasmids

Plasmid was extracted using the provided protocol (Quiagen Miniprep Kit).
The following plasmids were extracted: 203,104,101.

Plasmid was extracted using the provided protocol (JetStar Midiprep Kit).
The following plasmids were extracted: SDEN, SBEN, EDEN, lenti-CAS9-puro.

23.05.2017

Digestion of CAS9-GFP by BbsI

DNA from stock 2.
10 μl DNA [0.5ng/μl]
0.5 μl BbsI
2 μl 10x NEB2 buffer
5 μl H2O

Annealing of sgRNA oligos

5 μl 100mM of each Fwd and Rev ssOligos
10 μl 10x NEB2.1 buffer
80μl H2O
2 min. at 95°C cooling down in heatblock for 2h.

Testdigest of KO-Kit plasmids

Digest with EcoRV.
1.5 μl 10x Buffer
2 μl DNA
1 μl EcoRV
10,5 μl H2O
(Gelpicture-number:299)

Midiprep of SBEN, lenti-CAS9-puro

Plasmid was extracted using the provided protocol (JetStar Midiprep Kit).
The following plasmids were extracted: SBEN, lenti-CAS9-puro.

25.05.17

Testdigest of KO-KIT (101,104,203) with EcoRV

Volume	Component
1,5μl	Buffer
2μl	DNA
1μl	EcoRV
10,5μl	H2O

Digest at 37°C for 1h. Gel: undigested 203_2, 203_2, 203_1, 101_2, 101_1, 104_2, 104_1, ladder
Img: 300-303

Ligation of sgRNAs 1;3 into CAS9 GFP

No colonies from 24.05.17
Transformation with heatshock protocol of 10μl of remaining ligation from 24.05.17

26.05.17

Sitedirected mutagenesis (SDM) Q5 PCR with sgRNA overhang primers.

Test with sgRNA 1;20

Temp.[°C]	Time	Cycles
Volume	Component
12.5μl	Q5 2x Mastermix
1.25μl	10μM Fwd Primer
1.25μl	10μM Rev Primer
1μl	40 ng/μl CAS9-GFP
9μl	H2O
98	30 sec.
98	10 sec.
54/61	45 sec.
72	4.5 min.
98	10 sec.	24
64/70	30 sec.	24
72	3 min.	24
4	2 min.

Agarose Gel showed bands at wrong size (4kb)
Gelextraction
not further used

Ligation of sgRNA in CAS9-GFP

Few colonies observed
Plates thrown out because nonsense

27.05.17

Test of Midi Prep Kit

use of different Kits & performed at 2 Labs
similar concentrations observed

28.05.17

SDM PCR sgRNA1 CAS9GFP gradient

Volume	Component
12.5μl	Q5 2x Mastermix
1.25μl	10μM Fwd Primer
1.25μl	10μM Rev Primer
1μl	40 ng/μl CAS9-GFP
9μl	H2O

Program, gradient PCR:

Temp.[°C]	Time	Cycles
98	30 sec.
98	10 sec.
61/62/63/64	45 sec.
72	4.5 min.
98	10 sec.	24
70/71/72/73	6.5 min.	24
4	2 min.

Agarose gel: loading low temp → high temp
8kb band observed (expected 9kb)

29.05.17

SDM PCR for all sgRNAs CAS9-GFP

Volume	Component
12.5μl	Q5 2x Mastermix
1.25μl	10μM Fwd Primer
1.25μl	10μM Rev Primer
1μl	40 ng/μl CAS9-GFP
9μl	H2O

program: sgRNAs 1;5;20

Temp.[°C]	Time	Cycles
98	30 sec.
98	10 sec.
63	45 sec.
72	4.5 min.
98	10 sec.	24
72	6.5 min.	24
4	2 min.

program: sgRNAs 3;4;9

Temp.[°C]	Time	Cycles
98	30 sec.
98	10 sec.
59	45 sec.
72	4.5 min.
98	10 sec.	24
72	8 min.	24
4	2 min.

30.05.17

BsmBI digest of lenti-CAS9-puro (AG Cathomen)

20μl ~80ng/μl lenti-CAS9
5μl 10x NEB 3.1 Buffer
0.2μl BsmBI
24μl H2O

@55°C; 1h incubation.

SDM PCR of sgRNA1 vs. ctrl

Image: 315

31.05.17

SDM PCR for sgRNA5;20

Volume	Component
12.5μl	Q5 2x Mastermix
1.25μl	10μM Fwd Primer
1.25μl	10μM Rev Primer
1μl	40 ng/μl CAS9-GFP
9μl	H2O

program: sgRNAs 5;20

Temp.[°C]	Time	Cycles
98	30 sec.
98	10 sec.
59	45 sec.
72	4.5 min.
98	10 sec.	24
72	8 min.	24
4	2 min.

Mix 1: sgRNA 1;5;20		Mix 2: sgRNA 1;5;20		Mix 3: sgRNA1
volume	component	volume	component	volume	component
12.5μl	Q5 2x Mastermix	5μl	Q5 Buffer	12.5μl	Q5 2x Mastermix
1.25μl	10μM Fwd Primer	0.125μl	40mM dNTPs	1.25μl	10μM Fwd Primer
1.25μl	10μM Rev Primer	1.25μl	10μM Fwd Primer	1.25μl	10μM Rev Primer
1μl	40 ng/μl CAS9-GFP	1μl	400ng/μl CAS9-GFP	1μl	40 ng/μl CAS9-GFP
		0.25μl	Q5 Polymerase
9μl	H2O	16,125μl	H2O	9μl	H2O

Gelextraction of sgRNA1 in CAS9-GFP

BsmBI digest of lenti-CAS9-puro

volume	component
20μl	~70ng/μl plasmid
2.5μl	10x NEB 3.1
0.2μl	BsmBI
12.3μl	H2O

01.06.17

CAS9-GFP-sgRNA1 T4 ligation & DpnI digest

volume	component
4μl	Gel extract
1μl	T4 ligase
2μl	10x T4 Buffer
13μl	H2O

volume	component
2μl	Ligation
1μl	Cutsmart 10x Buffer
1μl	DpnI
6μl	H2O

Transformation of XL10gold with heatshock protocol

Ligated digest of CAS9-GFP-sgRNA1
pipette tip of CAS9-GFP stock

Maxi Prep Kit of KO-KIT plasmids

Zymo Maxi Prep Kit Zentrifugationsprotocol
200ml LB-Amp overnight culture

scramble plasmid: 144.3 ng/μl (400μl)
plasmid 203: 134.9 ng/μl (400μl)
plasmid 104: 115.0 ng/μl (400μl)

02.06.17

SMD PCR CAS9-GFP sgRNA 1;5;20;ctrl

protocol as previously; control with primers for sgRNA20

BsmBI digest of lenti-CAS9-puro

volume	component
20μl	~70ng/μl plasmid
2.5μl	10x NEB 3.1
0.2μl	BsmBI
12.3μl	H2O

samples taken at 2; 2.5; 3 hours

SDM PCR CAS9-GFP-sgRNA5

labbook weird O.o

Inoculation of 5ml culture LB-Amp with XL10gold

5 colonies sgRNA1
2 CAS9-GFP empty

03.06.17

BsmBI digest of lenti-CAS9-puro

volume	component
20μl	~70ng/μl plasmid
2.5μl	10x NEB 3.1
0.5μl	BsmBI
7μl	H2O

digest @ 55°C for 2h
10μl [50 ng/μl] plasmid loaded as control
Image: 333/334

Midi Prep

CAS9-GFP empty
CAS9-GFP sgRNA1

Production of glycerol stocks 1x CAS9-GFP; 5x CAS9-GFP-sgRNA1

SDM PCR of CAS9-GFP sgRNA5

Protocol same as 26.05.17
Dilution series of plasmid stock: 200ng/μl; 100ng/μl; 50ng/μl; 25ng/μl; 12.5ng/μl; 6.25ng/μl
New 1:10 dilution of primer stock was made

04.06.17

PCR eGFP-fragment for SGEN plasmid

volume	component
10μl	5x Q5 Buffer
1μl	10mM dNTPs
2.5μl	10mM FW primer (olG17_111)
2.5μl	10mM RW primer (olG17_110)
0.2μl	Template (plG_9)
0.5μl	Q5 Polymerase
33.3μl	H2O

Temp. [°C]	Time	Cycles
98	30 sec.
98	10 sec.	33
67	30 sec.	33
72	20 sec.	33
72	2 min.

PCR SDEN short fragment for SGEN plasmid

volume	component
10μl	5x Q5 Buffer
1μl	10mM dNTPs
2.5μl	10mM FW primer (olG17_109)
2.5μl	10mM RW primer (olG17_108)
0.2μl	Template (1754 base fragment of SGEN)
0.5μl	Q5 Polymerase
33.3μl	H2O

Temp. [°C]	Time	Cycles
98	30 sec.
98	10 sec.	33
67	30 sec.	33
72	20 sec.	33
72	2 min.

SDEN BamHI digest

volume	component
2μl	Plasmid (SDEN)
3μl	Cut Smart 10x Buffer
1μl	BamHI
24μl	H2O

Digest at 37°C for 1 hour. Expected fragment lenght: 4768 bp, 2386 bp.
Gel extraction of GFP-fragment. Obtained concentration: 7,8 ng/μl.

CAS9-GFP-sgRNA 5 Ligation

Mix 1: 200ng/μl template (12kb fragment)		Mix 2: 200ng/μl template (10kb fragment)		Mix 3: 50ng/μl template (10kb fragment)
volume	component	volume	component	volume	component
8μl	purified PCR product	8μl	purified PCR product	8μl	purified PCR product
2μl	T4 ligase 10x buffer	2μl	T4 ligase 10x buffer	2μl	T4 ligase 10x buffer
1μl	T4 ligase	1μl	T4 ligase	1μl	T4 ligase
9μl	H2O	9μl	H2O	9μl	H2O

Plasmids were ligated for 2 hours at 16°C.

Ligated plasmid was digested with DpnI for 1 hour at 37°C.

volume	component
4μl	Ligated plasmid
1μl	Cut Smart 10x buffer
1μl	DpnI
4μl	H2O

Oligocloning sgRNA1/3 in lenti-CAS9-puromycin

volume	component
7μl	11kb fragment of BsmBI digested lenti-CAS9-puromycin plasmid
1μl	50nM dilution of annealed dsOligo (sgRNA1/3)
1μl	T4 ligase 10x Buffer
1μl	T4 ligase

As a control 1μl H2O was used instead of dsOligos.

Transformation of XL10gold via heatshock transformation

3 Ligated & digested CAS9-GFP-sgRNA5 plasmid
sgRNA1/3/control of lenti-CAS9-puromycin plasmid

Heatshock transformation was performed accoarding to the heatshock protocol.

05.06.17

Digest of lenti-CAS9-puromycin

volume	component
15μl	Plasmid [c=70 ng/μl]
0,5μl	BsmBI
2,5μl	NEB 3.1 10x buffer
7μl	H2O

Digest for 2 hours at 55°C.
Gel: with EtBr.

SDM PCR for CAS9-GFP-shRNA3;4;9

volume	component
12,5μl	Q5 MasterMix 2x
1,25μl	FW primer (for sgRNA3,4,9)
1,25μl	RW primer (for sgRNA3,4,9)
1μl	CAS9-GFP plasmid
9μl	H2O

PCR program: sgRNA insertion 2.

step	temperature (°C)	time
1	98.0	00:30
2	98.0	00:10
3	57.8	00:45
4	70.8	06:30
5	98.0	00:10
6	70.8	07:00
7	72.0	10:00
8	72.0	pause

Steps 2 to 4 were repeated 1x and steps 5 to 6 were repeated 24x

06.06.17

Miniprep of overnight lenti-CAS9-sgRNA3

Miniprep was performed accoarding to the manufactors protocol.

SDM PCR for sgRNA3,4,9

volume	component
1μl	CAS9-GFP plasmid
0,5μl	FW primer (for sgRNA3,4,9)
0,5μl	RW primer (olG_118)
2,5μl	10x Taq buffer
0,5μl	dNTPs
1μl	Taq (peqlab)
19,875μl	H2O

Annealing at 54°C, Elongation 72°C for 40sec., 30 cycles.

07.06.17

Gelelectrophoresis of PCRs

SDM all failed.
Colony PCR positive for

sgRNA5 in CAS9-GFP
sgRNA in lenti-CAS9-puromycin

SDM PCR for CAS9-GFP-shRNA3,4,9

volume	component
12,5μl	Q5 MasterMix 2x
1,25μl	FW primer (for sgRNA3,4,9)
1,25μl	RW primer (for sgRNA3,4,9)
1μl	CAS9-GFP plasmid
9μl	H2O

PCR program: sgRNA insertion 2.

Gel: 363,364
Loading: sgRNA9, sgRNA4, sgRNA3

08.06.17

Colonie PCR for sgRNA 1,3,5

volume	component
1μl	Purified plasmid
0,5μl	FW primer (for sgRNA1,3,5)
0,5μl	RW primer (olG_118)
2,5μl	10x Taq buffer
0,5μl	dNTPs
0,125μl	Taq (peqlab)
19,875μl	H2O

Picture: 373

09.06.17

Digest of lenti-CAS9-puromycin with BsmBI

volume	component
15μl	lenti-CAS9-puromycin [c=70ng/μl]
0,5μl	BsmBI
2,5μl	NEB 3.1 10x Buffer
7μl	H2O

Digest for 2 hours at 55°C.

10.06.17

SDM PCR for CAS9-GFP-sgRNA3,4,9

volume	component
12,5μl	Q5 MasterMix 2x
1,25μl	FW primer (for sgRNA3,4,9)
1,25μl	RW primer (for sgRNA3,4,9)
1μl	CAS9-GFP plasmid
9μl	H2O

PCR program: sgRNA insertion 2.

Ligation of sgRNA 4,5,9,20 into lenti-CAS9-puromycin plasmid

volume	component
7μl	11kb fragment of BsmBI digested lenti-CAS9-puromycin plasmid
1μl	50nM dilution of annealed dsOligo (sgRNA4/5/9/20)
1μl	T4 ligase 10x Buffer
1μl	T4 ligase

As a control 1μl H2O was used instead of dsOligos.

Heatshock transformation of XL10gold

Heatshock transformation was performed accoarding to the heatshock protocol.

Digest of lenti-CAS9-puromycin with BsmBI

volume	component
15μl	lenti-CAS9-puromycin [c=70ng/μl]
0,5μl	BsmBI
2,5μl	NEB 3.1 10x Buffer
7μl	H2O

Digest for 2 hours at 55°C.

Agarose gel for CAS9-GFP-sgRNA3,4,9

Pic:

11.06.17

Minipreps of CAS9-GFP-sgRNA1, CAS9-GFP, lenti-CAS9-puromycin

Minipreps were performed accoarding to the manufacturer's protocol.

SDM PCR for CAS9-GFP-sgRNA3,4,9

volume	component
12,5μl	Q5 MasterMix 2x
1,25μl	FW primer (for sgRNA3,4,9)
1,25μl	RW primer (for sgRNA3,4,9)
1μl	CAS9-GFP plasmid
9μl	H2O

PCR program: sgRNA insertion 2.

12.06.17

Minipreps of lenti-CAS9-puro-sgRNA4,5,9,20

Minipreps were performed accoarding to the manufacturer's protocol.

Gelextraction of CAS9-GFP-sgRNA3,4,9

Gelextraction was performed accoarding to the manufacturer's protocol.

13.06.17

CAS9-GFP-sgRNA 5 Ligation

volume	component
8μl	purified PCR product
2μl	T4 ligase 10x buffer
1μl	T4 ligase
9μl	H2O

Plasmids were ligated for 2 hours at room temperature. Ligated plasmid was digested with DpnI for 1 hour at 37°C.

volume	component
4μl	Ligated plasmid
1μl	Cut Smart 10x buffer
1μl	DpnI
4μl	H2O

10μl ligated and digested plasmid was transformed using the heatshock protocol.

Heatshock transformation of XL10gold with CAS9-GFP-sgRNA3,4,9

Transformation of XL10gold was performed accoarding to the heatshock protocol.

14.06.17

SDM PCR for CAS9-GFP-sgRNA20

volume	component
12,5μl	Q5 MasterMix 2x
1,25μl	FW primer (for sgRNA20)
1,25μl	RW primer (olG_118)
1μl	CAS9-GFP plasmid
9μl	H2O

PCR program: sgRNA insertion 2.

15.06.17

Gelextraction of CAS9-GFP-sgRNA20

Gelextraction was performed accoarding to the manufacturer's protocol.

Gibson-Assambly

volume	component
1,8μl	SDEN backbone
1,4μl	SDEN fragment
1,8μl	GFP
5μl	assembly-mix

15 min. at 55°C
thaw Dh5α on ice
add 5μl Gibson-mix
incubate on ice for 10 min.
add 900μl LB-media
incubate for 1 hour at 37°C
spread on LB-Amp plate

16.06.17

CAS9-GFP-sgRNA20 Ligation

volume	component
8μl	purified PCR product
2μl	T4 ligase 10x buffer
1μl	T4 ligase
9μl	H2O

Plasmids were ligated for 2 hours at room temperature.

Restriction digest of SDEN-Backbone BamHI

volume	component
5μl	SDEN
2μl	CutSmart 10x Buffer
1μl	BamHI
12μl	H2O

Incubate for 2 hours at 37°C
Incubate for 20 min. at 65°C
Dephosphorylation:

volume	component
10μl	Antarctic phosphotase buffer
1μl	Antarctic Phosphotase

Incubation for 10 min. at 37°C
Gel purification (1% agarose, 115V, 30 min.)

Gibson-Assambly

volume	component
1,8μl	SDEN backbone
1,4μl	SDEN fragment
1,8μl	GFP
5μl	assembly-mix

15 min. at 55°C
thaw Dh5α on ice
add 5μl Gibson-mix
incubate on ice for 10 min.
add 900μl LB-media
incubate for 1 hour at 37°C
spread on LB-Amp plate

17.06.17

Colony PCR

for plasmid CAS9-GFP-sgRNA3/9
lenti-CAS9-puro-sgRNA1/3
positive control RNA5_col3

Annealing changed to 58°C

digest of CAS9-GFP-sgRNA20 with DpnI

volume	component
4μl	Ligation
1μl	Cut Smart 10x buffer
1μl	DpnI
4μl	H2O

Digest for 1 hour at 27°C. 10μl were transformed into XL10gold using the heatshock protocol.

Annealing of sgRNA oligos

volume	component
5μl	100mM of each Fwd and Rev ssOligos
10μl	10x NEB2.1 buffer
80μl	H2O

2 min. at 95°C cooling down in heatblock for 2h.

BsmBI digest of lenti-CAS9-puromycin

volume	component
1μl	lenti-CAS9-puromycin
1,5μl	10x NEB 3.1 buffer
0,5μl	BsmBI
12μl	H2O

Digest for 2 hours at 55°C.

Miniprep of SGEN-Gibson Colony

Minipreps were performed accoarding to the manufacturer's protocol.
Testdigest with NotI not succesful.

18.06.17

Ligation of sgRNA scramble into lenti-CAS9-puromycin

volume	component
7μl	11kb fragment of BsmBI digested lenti-CAS9-puromycin plasmid
1μl	50nM dilution of annealed dsOligo (sgRNAscramble)
1μl	T4 ligase 10x Buffer
1μl	T4 ligase

10μl were used for heatshock transformation.

Heatshock transformation of XL10gold

Heatshock transformation was performed accoarding to the heatshock protocol.

19.06.17

Miniprep of 7 SGEN-Gibson colonies

Minipreps were performed accoarding to the manufacturer's protocol.
Positive colonies were observed for colony 1,4,5 and 7.

SDM PCR for CAS9-GFP-sgRNAscramble

volume	component
12,5μl	Q5 MasterMix 2x
1,25μl	FW primer (for sgRNAscramble)
1,25μl	RW primer (olG_118)
1μl	CAS9-GFP plasmid
9μl	H2O

PCR program: sgRNA insertion 2.

10μl were used for heatshock transformation.

Heatshock transformation of XL10gold

Heatshock transformation was performed accoarding to the heatshock protocol.

20.06.17

Test digest of SGEN-Gibson clolonies

volume	component
x	80ng DNA
0,2μl	NotI
2μl	Cut Smart 10x buffer
to 20μl	H2O

Digest for 45 minutes at 45°C.

Heatshock transformation of colonies 1,2,4,5,7.

Heatshock transformation of Dh5α

Heatshock transformation was performed accoarding to the heatshock protocol.

21.06.17

digest of CAS9-GFP-sgRNA4,3,9 with DpnI

volume	component
4μl	Ligation
1μl	Cut Smart 10x buffer
1μl	DpnI
4μl	H2O

Digest for 1 hour at 37°C.
10μl was used for transformation.

Heatshock transformation of XL10gold

Heatshock transformation was performed accoarding to the heatshock protocol.

21.06.17

Sequencing

Plasmid	Sequencing Primer
SGEN 1	Seq SBEN 2
SGEN 2	Seq SBEN 2
SGEN 3	Seq SBEN 2
SGEN 4	Seq SBEN 2
SGEN 5	Seq SBEN 2
CAS9-GFP-sgRNA20 1	olG_117
CAS9-GFP-sgRNA20 2	olG_117
CAS9-GFP-sgRNA3 1	olG_117
CAS9-GFP-sgRNA9 1	olG_117
lenti-CAS-sgRNA1	olG_117
lenti-CAS-sgRNA3	olG_117

22.06.17

SDM PCR for shRNA Inserts

volume	component
10μl	Template
10μl	Q5 5x buffer
1μl	10μM dNTPs
2,5μl	FW primer
2,5μl	RW primer
0,5μl	Q5 polymerase
23,5μl	H20

Temp.[°C]	Time	Cycles
98	30 sec.
98	10 sec.	33
66	30 sec.	33
72	20 sec.	33
72	2 min.	24

24.06.17

overnight culture

Overnight culture was prepared from glycerol stocks. Plasmid 101, 104,203 of Origene KO-Kit.

25.06.17

overnight culture

100ml for Midi prep was prepared for plasmid 101, 104,203 of Origene KO-Kit.

26.06.17

Midi prep

Midi prep for plasmid 101, 104,203 of Origene KO-Kit was performed accoarding to the manufacturer's protocol.

27.06.17

Heatshock transformation

Heatshock transformation of KO-Kit scramble sgRNA was performed accoarding to protocol.

Colony PCR of lenti-CAS9-puro plasmids

volume	component
1 μl	Template
2 μl	10x Buffer
0.4 μl	10 μM dNTPs
1 μl	FW primer
1 μl	RW primer
0.25 μl	Taq polymerase
14.35 μl	H20

Expected bands: 2,804 bp

Overnight digest of lenti-Cas9

volume	component
2.5 μl	Plasmid (500ng/µl)
1 µl	BsmBI
5 µl	3.1 10x buffer
41.5 µl	H2O

Digest overnight at 55°C.

Picture nr. 468

Colony PCR of Cas9-GFP sgRNA1/4/scramble and lenti-Cas9 sgRNA5

Colony PCR was performed according to protocol.
For Cas9-GFP: forward primer was singlestrand oligo forward as used to produce double strand oligos for cloning, revese primer 118.
For lenti-Cas9: primers 117 and 118.
Annealing at 58°C.

28.06.17

Digest of lenti-Cas9 with BsmBI

volume	component
1 μl	Template
2 μl	10x Buffer
0.4 μl	10 μM dNTPs
1 μl	FW primer
1 μl	RW primer
0.25 μl	Taq polymerase
14.35 μl	H20

Expected bands: 2,804 bp

Picture no. 465

Overnight digest of lenti-Cas9

volume	component
2.5 μl	Plasmid (500ng/µl)
1 µl	BsmBI
5 µl	3.1 10x buffer
41.5 µl	H2O

Digested for 2h at 55°C.
Gel overnight 2h undigested, picture nr. 468

Miniprep of lenti-Cas9

Miniprep was performed according to the manufacturer's protocol.

PCR of knockdown inserts

Templates (97meres): olG17_112 to olG17_116.

volume	component
10.0 µl	5x Q5Bfr
1.0 µl	dNTPs (10mM)
2.5 µl	5'mirEXhoIfw (10 µM)
2.5 µl	3'mirEEcoRIrv (10 µM)
2.0 µl	template (0.01 ng/µl)
0.5 µl	Q5 Pol
31.5 µl	H2O

PCR program as described on 22.06.17.
Purification with PCR purification kit according to the manufacturer's protocol (Qiagen), elution in 30 µl H2O.

Digest of lenti-Cas9 with BsmbI

volume	component
2.5 μl	Plasmid (500ng/µl)
1 µl	BsmBI
5 µl	3.1 10x buffer
41.5 µl	H2O

Digested at 55°C for 2 h.

volume	component
25 μl	Plasmid (500ng/µl)
1 µl	BsmBI
5 µl	3.1 10x buffer
19 µl	H2O

Digested at 55°C overnight.

volume	component
2.5 μl	Plasmid (500ng/µl)
1 µl	BsmBI
5 µl	3.1 10x buffer
41.5 µl	H2O

Digested at 37°C overnight.

29.06.17

Digest of lenti-Cas9 with BamBI

volume	component
2.5 μl	Plasmid (500ng/µl)
1 µl	BsmBI
5 µl	3.1 10x buffer
41.5 µl	H2O

Digested at 55°C for 2 h.

Miniprep of lenti-CAS9

No. 1 grew at 30°C, No. 2&3 at 37°C.
Miniprep was performed according to manufacturer's protocol.
1 µl of mini was loaded on gel (picture no. 473).

30.06.17

Gel extraction of digested lenti-Cas9

Gel extraction was performed according to manufacturer's protocol.

01.07.17

Dephosphorylation of backbone

Was performed according to manufacturer's protocol (Fast AP).

Ligating sgRNA into lenti-Cas9 backbone

Annealed sgRNA oligos: 1, 3, 4, 5, 9, 20

volume	component
7 µl	backbone (9.3 ng/µl)
1 µl	annealed sgRNA oligo (50 nM)
1 µl	10x T4 buffer
1µl	T4

Transformation of ligation

sgRNAs: 1, 3, 4, 5, 9, 20 & scramble of KO kit Transformation was performed according to heat shock protocol.
–> Only colonies containing sgRNAscramble were obtained.

02.07.17

Digest of lenti-Cas9 with BSmbI

volume	component
2.5 μl	Plasmid (500ng/µl)
1 µl	BsmBI
5 µl	3.1 10x buffer
41.5 µl	H2O

Digested at 55°C for 2 h.

Ligating shRNA into lenti-Cas9 backbone

sgRNAs: 1, 3, 4, 5, 9, 20

volume	component
7 µl	backbone (9.3 ng/µl)
1 µl	annealed sgRNA oligo (50 nM)
1 µl	10x T4 buffer
1µl	T4

Ligation was performed at 16°C overnight.

03.07.17

Transformation of ligation

sgRNAscramble served as control.
Transformation was performed according to heat shock protocol.

04.07.17

Gel extraction and ligation

volume	component
12 µl	backbone
1 µl	sgRNA 1 & 9
1 µl	10x T4 buffer
1µl	T4

Ligation was performed 37°C for 2 h.

Digest of lenti-Cas9 with BsmBI

Digest reaction a)

volume	component
2.5 μl	Plasmid (500ng/µl)
1 µl	BsmBI
5 µl	3.1 10x buffer
41.5 µl	H2O

Digest reaction b)

volume	component
15 μl	Plasmid (100ng/µl)
1 µl	BsmBI
5 µl	3.1 10x buffer
29 µl	H2O

05.07.17

Colony for miniprep

5µl LB containing bacteria from colonies lenti-Cas9 c1 and c2 were incubated overnight.

Digest of lenti-Cas9 with BsmBI

volume	component
20 μl	Plasmid (30ng/µl - 50 ng/µl)
1 µl	BsmBI
5 µl	3.1 10x buffer
24 µl	H2O

07.07.17

Digest of lenti-Cas9 with BsmBI

volume	component
2 μl	Plasmid (250 ng/µl, prepped the day before)
2 µl	BsmBI
2.5 µl	3.1 10x buffer
18.5 µl	H2O

Digest was performed at 55°C for 2 h.

DpnI digest of Cas9-GFP ligations

sgRNA1 (PCR on 30.05.17, ligation on 01.06.17)
sgRNA4 (PCR on 10.06.17, ligation on 13.06.17)
sgRNA9 (PCR on 10.06.17, ligation on 13.06.17)
sgRNA20 (PCR on 14.06.17, ligation on 16.06.17)

Digest reaction a) for each sgRNA

volume	component
1 μl	DpnI
1 µl	CutSmart
2 µl	ligation
6 µl	H2O

Digest reaction b) for each sgRNA

volume	component
1 μl	DpnI
1 µl	CutSmart
4 µl	ligation
4 µl	H2O

Transformation into XL10gold performed according to heat shock protocol (06.05.17).

Test digest of Cas9-GFP sgRNA1 and sgRNA9

Cas9-GFP sgRNA1 colonies (PCR on 30.05.17)

volume	component
0.5 μl	NdeI
x µl	Cas0-GFP sgRNA1 plasmid (1µg)
1 µl	CutSmart bufffer
8.5-x µl	H2O

Cas9-GFP sgRNA9 colonies (PCR on 10.06.17)

volume	component
0.25 μl	NcoI
x µl	Cas0-GFP sgRNA1 plasmid (1µg)
1 µl	CutSmart bufffer
8.75-x µl	H2O

Heatshock transformation of Cas9-GFP plasmids

Transformation of Cas9-GFP sgRNA3 colonies 3/6 and sgRNAscramble colonies 2/4 into XL10gold was performed according to heatshock protocol.

08.07.17

Test digest of sgRNA3 and sgRNA scramble

a	b	c	d	component
1 µl	1 µl	2 µl	2 µl	NdeI
4 µl	-	4 µl	-	sgRNA c4 (250 ng/µl)
-	8 µl	-	8 µl	scramble c3 (250 ng/µl)
1 µl	1 µl	1 µl	1 µl	CutSmart
4 µl	-	4 µl	-	H2O

Digest was performed at 37°C for 2 h.

Colonies picked

Colonies were picked for sgRNA4 and sgRNA1 (5 colonies each) and sgRNA9 (2 colonies). Since no colony grew for sgRNA 20, the plate was incubated a second night. One picked for each re-trafo.

09.07.17

Test digest of 5 colonies sgRNA1 in Cas9-GFP

volume	component
x µl	plasmid (1 µg)
1 µl	NdeI
1 µl	CutSmart
8-x µl	H2O

Digest was performed at 37°C for 2 h.
sgRNA scramble, colony 3 negative control, colony 4 positive control.

Re-trafos sgRNA3/scramble

From overnight cultures of sgRNA3 and sgRNAscramble re-trafos 2 glycerol stocks, 1 miniprep (5.5 ml) and 1 overnight culture for midiprep (100ml made with 4 ml cultured bacter9a) were made for each sgRNA.
From overnight cultures of sgRNA1, sgRNA4 and sgRNA9 re-trafos 4 glycerol stocks and 4 minipreps were made for all sgRNAs.

10.07.17

Restriction digest of knockdown backbone SDEN

volume	component
6.3 µl	SGEN backbone (5 µg)
2 µl	10x CutSmart
1 µl	EcoRI
1 µl	XhoI
9.7 µl	H2O

Digest was performed for 2 h at 37°C.
Inactivation took 20 min at 65°C.

Dephosphorylation

volume	component
10 µl	antarctic phosphatase buffer
1 µl	antarctic phosphatase
10 µl	digested plasmid

Dephosphorylation took 10 min at 37°C.

Restriction digest of PCR fragments of knockdown inserts

GelEx (22.06.17)	PCREx (27.06.17)	component
9.1 ng/µl	11.0 ng/µl	olG17_112
4.5 ng/µl	5.7 ng/µl	olG17_113
8.9 ng/µl	5.6 ng/µl	olG17_114
11.3 ng/µl	7.9 ng/µl	olG17_115
5.9 ng/µl	6.3 ng/µl	olG17_116

All 10 samples were digested completely.

volume	component
25 µl	PCR-Fragments
0.5 µl	10x CutSmart
0.25 µl	XhoI
0.25 µl	EcoRI
19.5 µl	H2O

Digest was performed for 3 h at 37°C.
Inactivation took 20 min at 65°C.
Fragments were separated using gel electrophoresis (2% agarose, 120 V, 30 min).
The digested fragments were not visible in the gel, the phosphorylated backbone smeared (Picture: IM.000500.Tif). Only the non-dephosphorylated was backbone cut out and extracted from gel using a manufacturer's protocol.

Midiprep Cas9-GFP sgRNA3(c6)/scramble(c4)

Midiprep was performed according to manufacturer's protocol to obtain plasmids for electroporation from sequenced colonies (150 µg).

Site-directed mutagenesis PCR CAS9-GFP sgRNA3 & sgRNA20

PCR was performed with Q5 Master Mix for sgRNA3 and self-mixed solution for sgRNA 20.

11.07.17

PCR of 97meres for knockdown inserts

volume	component
10 µl	5x Q5 buffer
1 µl	dNTPs (10 mM)
2.5 µl	5'mirEXhoIfwd (10 µM)
2.5 µl	3'mirEEcoRIrev (10 µM)
2 µl	97mere template (0.01 ng/µl)
0.5 µl	Q5 Pol
31.5 µl	H2O

Templates: 0lG17_112 to olG17_116

step	temperature	duration
1	98°C	1 min
2	98°C	10 sec
3	66°C	30 sec
4	72°C	20 sec
5	72°C	2 min
6	8°C

Steps 2 to 4 were repeated 33x.
Expected product of 144 bp was observed (picture no. IM000506.Tif) and extracted from gel with Qiagen PCR extraction kit according to manufacturer's protocol and eluted in 30 µl H2O.

volume	component
5 µl	10x CutSmart buffer
0.25 µl	XhoI
0.25 µl	EcoRI
4.5 µl	H2O
40 µl	template

Previously eluted product was digested for 3 h at 37°C (10 µ from each PCR run were mixed, for each of the 5 templates). Fragments were separated using gel electrophoresis (2% agarose gel, 120 V, 20 min) but gel was empty apart from ladder (picture no. IM000504.Tif).

Troubleshooting showed that PCR extraction had not been successful (gel 120 V, 20 min, picture IM000510.Tif).

Digest SGEN backbone of Gibson colonies 7 and 2

volume	component
6.3 µl	SGEN plasmid (5 µg)
2 µl	CutSmart buffer
1 µl	EcoRI
1 µl	XhoI
9.7 µl	H2O

Digest was performed for 2 h at 37 °C.
Fragments were separated using gel electrophoresis (picture no. IM000502.Tif) and extracted with qiagen gel extraction kit according to manufacturer's protocol.

12.07.17

Overnight cultures of KO-Kit plasmids and lenti-Cas9

Overnight cultures of KO-Kit 101, 104, 203, lenti-Cas9 for minipreps and midipreps were prepared (13.07./14.07.).

Gels for checking purified knockdown insert DNA

No DNA was visible on gel since concentrations were too low (pictures no. IM000512.Tif and IM000514.Tif).

Purification, restriction digest and ligation of knockdown insert PCR products

Purification of PCR products of all 5 PCRs (picture IM000520.Tif) was performed using Qiagen gel extraction kit according to manufacturer's protocol. Samples washed twice with PE buffer show better curve on nanodrop.

Restriction digest of purified samples (samples from gel using 20x blue loading dye)

volume	component
17 µl	purified DNA
2 µl	10x CutSmart buffer
0.5 µl	EcoRI
0.5 µl	XhoI

2h at 37°C
Fragments were purified using Qiagen purification kit according to maufacturer's protocol.

ligation

volume	component
0.7 µl	digested backbone (50 ng)
x µl	digested insert (2,415 ng)
1 µl	T4 ligase buffer
1 µl	T4 ligase
7.3-x µ	H2O

Gel extraction of sgRNA3 inserted by SDM PCR into CAS9-GFP

Site-directed mutagenesis PCR of sgRNA20 into Cas9-GFP was repeated and failed.
2 overnight cultures of Cas9-GFP-sgRNA5 were prepared in order to obtain DNA for electroporation. Electrophoresis on agarose of miniprepped coloniees confirm that CutSmart was contaminated with DNAse but not the DNA samples (clear bands were observed as expected).

13.07.17

Sequencing sgRNAs in Cas9-GFP

DNA from colonies of original trafo with CAS9-GFP sgRNAs 1, 4 and 9, which were not yet sequenced or where sequencing hat previously failed, were send to sequencing.
Minipreps(2x) of sgRNA5 in Cas9-GFP were performed according to manufacturer's protocol for 2 electroporations

Heat shock with ligated knockdown plasmids

XL10gold bacteria were transformed with ligation sample (5 µl) following the heat shock protocol (5 plates containing plasmids based on olG17_112, olG17_113, olG17_114, olG17_115, olG17_116) Heat shock failed (bacteria were previously stored overnight at -20°C instead of -80°C, only 40 µl bacteria solution available per heat shock).

15.07.17

Overnight culture of XL10gold

An overnight culture of XL10 gold was prepared using 4 ml LB.

16.07.17

overnight culture of XL10gold

Overnight culture of XL10gold was enlagred to 50 ml LB.
ZymoBroth start: 20:00-9:30

17.07.17

Culturing XL10gold

Start value OD600 = 0.079
Using 1:10 dilution –> OD600 = 0.79

Digest Cas9-GFP

volume	components
0.5 µl	Cas9-GFP (1.1 µg/µl, 11.06.17)
0.25 µl	EcoRI
0.25 µl	NotI
1 µl	CutSmart
8 µl	H2O

Expected fragments: 3300 bp, 60000 bp (picture no. 529)
Fragments were observed and 3.3 kb fragment was excised for extraction.

Heatshock transformation of knockdown ligations and lenti-CAS9

Transformation with knockdown ligations 112, 113, 114, 115, 116 and lenti-Cas9 1516 were performed according to heatshock protocol.

Gel purification protocol

add 450 µl QG buffer, 50-60°C for 10 min
add 150 µl isopropanol and mix
10 min at -20°C
load on column
spin for 1 min at full speed and remove flow through
wash with 750 µl PE buffer
spin for 1 min at full speed and remove flow through
spin 2 min at full speed and remove flow through
elute DNA in 30 µl H2O

19.07.17

Ligation of sgRNA3 in Cas9-GFP SDM PCR product

volume	component
4 µl	8 gn/µl PCR product
4 µl	H2O
1 µl	T4 ligase
1 µl	ligase buffer 10x

3 samples 2 h at 16°C
3 samples 1 h at 16°C
DpnI digest (4 µl of each ligation) and heat shock frafo in XL10gold

Minipreps of picked knockdown cultures

Minipreps of knockdown plasmids 112, 113, 114, 115, 116 were performed according to manufacturer's protocol and send for sequencing.

20.07.17

Picking colonies of CAS9-GFP sgRNA3

One colony of each plate of CAS9-GFP sgRNA3 was picked for prep, glycerol stock and digest. Plates were stored at 4°C to pick further colonies (re-plate tomorrow).

21.07.17

Extraction of genomic DNA from HEK293T cells

Genomic DNA was extracted from HEK293T cells according to kit protocol for T7E1 assay establishment.

Positive knockdown colonies

Positive colonies of 114, 115, 116 (SGEN) were recultured and new colonies of 112, and 113 were picked.

Miniprep of lenti-CAS9 colonies

A miniprep of 5 lenti-CAS colonies was performed according to manufacturer's protocol.

lenti-Cas9 test digest with NcoI-HF was performed at 37°C.

volume	component
2 µl	DNA
0.25 µl	NcoI-HF
1 µl	10x CutSmart
6.75 µl	H2O

picture no. 534

Digest of lenti-Cas9 was performed using BsmbI.

volume	component
2.5 µl	plasmid (500 ng/µl)
5 µl	fast digest buffer
1 µl	BsmBI
41.5 µ	H2O

picture no. 534

Miniprep of new picked knockdown colonies

Miniprep was performed of knockdown colonies 112 and 113 according to manufacturer's protocol.

24.07.17

Digest of lenti-Cas9

volume	component
6 µl	plasmid (279.5 ng/µl)
5 µl	fast digest buffer 10x
1 µl	BsmBI
38 µl	H2O

Site directed mutagenesis PCR

Site directed mutagenesis PCR was perfored to obtain BsmBI and BbsI restriction sites in Cas9-GFP and BbsI in Lenti-Cas9-puro. Two fragments were generated for each insertion.

Fragment		1		2
Plasmid	Restriction site	primers	size	primers	size
GFP	BsmBI	212/215	3 kb	216/218	3.7 kb
GFP	BbsI	212/214	3 kb	216/218	3.7 kb
lenti	BbsI	213/214	1 kb	216/217	2.9 kb

The observed fragment pattern was observed ladder was not visible on gel. Fragments were excited anyway and PCR was repeated on 25.07.17.

25.07.17

Gel extraction, digest and ligation of lenti-Cas9

lenti-CAS9 fragments were extracted from gel and digested using BsmBI.

volume	component
6 µl	plasmid
5 µl	FD 10x B.
1 µl	BsmBI
38 µl	H2O

Picture no. 543

Fragments of lenti-Cas9 were ligated.

volume	component
5 µl	backbone (15.9 ng/µl)
1 µl	sgRNA1, 3, 4 (50 nM)
1 µl	T4 buffer
1 µl	T4
2 µl	H2O

T7E1 assay

NEB 2 puffer 1x and 1.5 µl T7 were added to 200 ng of PCR products (of positive control, exon 6, exon 5, exon 8, exon 10). Assay was cooled down in 2.5 h from 95°C to 20°C.

Miniprep of SGEN 115 and lenti-Cas9 plasmids

Minipreps were performed according to manufacturer's protocol.

26.07.17

Picking colonies for lenti-Cas9

Colonies containing sgRNA1, 3, 4, 5, 9 in lenti-CAS9 were picked.

Digest of lenti-Cas9 with BsmBI

volume	component
3 µl	plasmid
2.5 µl	FD buffer
0.5 µl	BsmBI
19 µl	H2O

picture no. 547

SDM PCR BsmBI and BbsI into Cas9-GFP and lenti-Cas9-puro

Site-directed mutagenesis was performed as previously to insert BsmBI and BbsI into Cas9-GFP. PCR fragments were excised from gel and purified using a clear-up kit according to manufacturer's protocol. Fragments were digested with BsmBI or BbsI and NotI or EcoRI and purified using a PCR clear-up kit.
Cas9-GFP and lenti-Cas9-puro were digested with NotI and EcoRI in FD 10x.
picutres no. 539 and 538

27.07.17

Site directed mutagenesis PCR

Strategy was changed because strategy with shorter fragments for each side combined with NotI and EcoRI did not work (produced blunt-ends). Now a whole plasmid amplification was performed to insert BsmBI and BbsI into Cas9-GFP (run on 0.9% agarose at 110 V for 35 min, picture no. IMG00550). Protocols as for oroginal blunt end ligation estrategy, expected band of 9.3 kb was at 10 kb with good efficiency, also a 3 kb band was observed and both were extracted.

Digest with BsmBI or BbsI

volume	component
0.5 µl	BsmBI
	fermentas FD 10x
1 µl	DpnI
	H2O

Digest was performed for 20 min at 37 °C and 1 h at 55°C.

volume	component
1 µl	BbsI
	fermentas FD 10x
1 µl	DpnI
	H2O

Digest was performed for 2 h at 37°C.

Oligo ligation of sgRNA5 with T4 was performed for 1 h and XL10gold were transformed according to heat shock protocol. Colonies were also found in BbsI (maybe confused) and BsmpI (DpnI might need longer).

Miniprep of Lenti-Cas9

Minipreps of plasmids containing sgRNA1, 3, 4, 5, 9 were performed according to manufacturer's protocol and send for sequencing.

28.07.17

Gelex of digested lenti-CAS9 and ligation of lenti-Cas sgRNA20

volume	component
7 µl	plasmid
1 µl	sgRNA (50 ng/µl)
1 µl	T4 ligase buffer
1 µl	T4 ligase

PCR for T7E1

PCR for T7E1 was performed to identify knockout in HEK cells as previously described. PCR purification was performed of HEK both primers and exon 8.

30.07.17

PCR for T7E1 assay

Positive control: CCR5delta32 mutation in HEK293T (olG17_073/074).
Negative sample: HIF1A exon 8.

volume	component
x µl	gDNA (300ng)
5 µl	5x phusion HF buffer
0.5 µl	dNTPs
1 µl	fwd
1 µl	rev
0.25 µl	phusion
17.25-x µl	H2O

(original protocol: 200 ng gDNA)
Better use 0.5 µl primers for 25 µl reactions.
Electrophoresis was performed on 2% agarose gel (100 V for 45 min).
Expected bands: 500 bp

PCR HEK positive control 73,74 test

volume	component
8 µl	H2O
12,5 µl	kappa2G
1.25 µl	primer 73
1.25 µl	primer 74

31.07.17

Sequencing of Cas9-GFP sgRNAs

Cas9-GFP sgRNAs1, 3, 4, 9, 20 from SDM PCR restriction site insertion, BsmBI or BbsI and DpnI digested and dsDNA oligoinsertion were send to sequencing.

PCR purification of T7E1 samples

T7E1 dsDNA samples obtained on 30.07.17 were dissociated and re-annealed in NEB 2 buffer 1x at 95°C for 5 min and for 2:30 h slowly cooled down to room temperature in heatblock.

T7E1 assay

200 ng PCR product were re-annealed using 1.5 µl 1:1 of T7E1 and and 2x NEB 2 buffer at 37°C for less than 20 min. Reaction was stopped by addition of 3 µl 6x OrangeDye, the samples were put on ice immediately and quickly loaded on 2% agarose gel.
Expected fragments: 200 bp, 300 bp, could not be observed.

01.08.17

Knockout Sequencing

Cas9-GFP sgRNAs from 31.07.17, only positive for BsmBI samples
repeat of SDM PCR with Q5 to insert BsmBI sites into Cas9-GFP
sequencing of other colonies for sgRNAs 4, 9 inserted by BsmBI, overnight cultures for miniprep

T7E1 assay repeat troubleshooting

use more DNA
anneal longer than 3 h
T7 less than 15 min

T7E1 PCR

2x at 63°C, no good result on first gel

02.08.17

Miniprep of Cas9-GFP sgRNA4/9

2 more colonies for each BsmBI cloning (27.07.17) because sequencing was negative
picking 3 colonies from BsmBI cloning (01.08.17) for sgRNAs3/20 in Cas9-GFP, other plates stored
control transformation yielded no colonies this time, maybe because of longer digestion with DpnI
5 ml overnight cultures (2x each)

lenti-Cas9-puro sgRNas 1 / 3 / 4 at 37°C
SGEN shRNA 4 (former 115, now 133) and shRNA 5 (former 116, now 134) at 30°C

T7E1 PCR

PCR at 60°C worked with HEK but not wich Ex8.
PCR purification HEK (30 ng) and Ex8 (15 ng).

Standard PCR protocol HEK 73/74

volume	component
0.5 µl	DNA (200 ng)
5 µl	phusion 5x buffer
0.5 µl	dNTPs
0.5 µl	fwd
0.5 µl	rev
0.25 µl	phusion
17.75 µl	H2O

03.08.17

T7E1 annealing

HEK CCR6 PCR product, 1 h,2 h, and 6 h at 37°C, no negative control
T7E1 incubation for 29°C at 37°C

Minipreps and Glycerol stocks

low yield minipreps
sgRNA1, 3, 4 in Lenti-Cas9
sgRNA3, 20 in Cas9-GFP
sgRNA1 in Cas9-GFP
new overnight cultures 9 ml Cas9-GFP sgRNA1, 4, 5, 2 colonies of Cas9
miniprep (promega) of lenti-Cas9 colonies

04.08.17

PCR on gDNA for T7

New primers for exon 9, 10 in Hif1A improved PCR, old primers for exon 6, 8 worked fine. Annealing at 60°C annealing lead to weak band for exon 6. No bands were observed for CCR5delta 32 in HEK (bad gDNA was used, now thrown away). Experiment for CCRDdelta32 was repeated with good gDNA(59°C annealing) to obtain new samples.

PCR on gDNA of Jurkat cells with KO-Kit primers

volume		component
0.2 µl	1.8 µl	Taq
1.5 µl	13.5 µl	buffer
1 µl	9 µl	template
0.3 µl	2.7 µl	dNTPs
0.75 µl	6.75 µl	fw
0.75 µl	6.75 µl	rv
10.5 µl	95.5 µl	H2O

PCR gDNA in was annealed in thermocycler.

95-85°C at 2°C/min
85-25°C at 0.05°C/min

T7E1 endonuclease assay

In Jurkat cells 200 ng gDNA were annealed (HIF1A 1.5 µl 1:1 T7:NEB2 2x, exon 6). No control was used.
In HEK293T cells 200 ng gDNA were annealed (CCR5delta32 1.5, 2, 2.5, 3, 4 used 1:1 T7:NEB2). Control was used.
Annealing was performed at 37°C for 25 min.

Minipreps Cas9-GFP

Minipreps of Cas9-GFP sgRNA1, 4, 5 were performed for electroporation according to manufacturer's protocol.

05.08.17

T7E1 annealing

HEK

volume	component
4 µl	DNA (200ng)
0.75 µl	T7
0.75 µl	NEB2 2x
8 µl	NEB2 1x

Positive control

volume	component
4 µl	gDNA (58 ng/µl)
0.75 µl	T7
0.75 µl	NEB2 2x
8 µl	NEB2 1x

Negative control

volume	component
4 µl	gDNA exon 5 (25 ng/µl)
0.75 µl	T7
0.75 µl	NEB2 2x
8 µl	NEB2 1x

Gel at 95V for 50 min.

PCR KO-Kit confirm

PCR was performed to confirm knockout.

volume	component
0.2 µl	taq
2 µl	template
1.5 µl	10x buffer
0.3 µl	dNTPs
0.75 µl	fw (32)
0.75 µl	rv (59)
9.5 µl	H2O

Glycerol stocks Cas9-GFP

Cas9-GFP BsmbI

sgRNA20 colonies 4, 5 and 6
sgRNA3 colonies 4, 5 and 6

Test digest of SGEN112_2_1 and SGEN 112_2_2

volume	component
2 µl	DNA
1 µl	10x FD
0.5 µl	NotI
6.5 µl	H2O

Digest was performed at 37°C for 1 h.
Picture in labbook\

Minipreps of Cas9-GFP sgRNA3 and sgRNA20

3 more colonies for Cas9-GFP sgRNA3 and sgRNA20 were sent for sequencing.
Overnight cultures (5 ml LLB-Amp)

for electroporation lenti-Cas9-puro sequenced sgRNAs1-9
for cloning constitutively active (CCa) HIF1A as western blot positive contro, 1x HA-HIF1A from plate

07.08.17

Minipreps of 101 and 104

Minipreps were performed according to manufacturer's protocol.

Positive sequencing result for Cas9-GFP-sgRNA9

Colony 4 of Cas9-GFP-sgRNA9 was positive. Cas9-GFP sgRNA3 and sgRNA20 were send for sequencing (3 colonies each).

08.08.17

Miniprep of KO-Kit

Miniprep of plasmids 101, 104, 203 was performed according to manufacturer's protocol.

Positive sequencing and picking colonies

Sequencing was positive for Cas9-GFP sgRNa3 (colony 5). 4 new colonies of Cas9-GFP sgRNA20 were picked (LB-Amp 5ml overnight cultures). Cultures of Cas9-GFP sgRNA3, sgRNA9 for preps for electroporation and SGEN shRNA112 for lentisviral transduction.

T7E1 assay

T7E1 assay was performed.

PCR 4x HEK293T CCR5delta32 positive control
PCR 1x each of Jurkat HIF1A exons 5, 6, 8, 10 negative control
PCR clearup from 2% agarose gel (Qiagen kit)
annealing at 95-85% 2°C/sec, 85-25°C 1% ramps(approx. 0.6°C/sec)
assay 12 µl DNA + 1.5 µl 1xNEB2

Tested conditions

	+ T7E1								no T7E1
	pos. ctrl.	pos. ctrl.	pos. ctrl.	pos. ctrl.	pos. ctrl.	pos. ctrl.	pos. ctrl.	neg. ctrl.	pos. ctrl.	neg. ctrl.
Method	Cycler	Cycler	Block	Water	Cycler	Cycler	Cycler	Cycler	room temp	room temp.
Amount DNA	200	200	200	200	200	300	400	200	200	200
Duration	10	15	20	20	20	20	20	20	20	20

KO-Confirm PCR with primers 228 and 229

PCR was performed on plasmid 104 and gDNA to confirm knockout.

volume	component
2 µl	template
1.5 µl	10x buffer
0.3 µl	dNTPs
0.75 µl	primer fw
0.75 µl	primer rv
0.2 µl	Taq
9.5 µl	H2O

Program koconfirm (but here at 64°C)

step	temperature (°C)	time
1	95	3:00
2	95	0:30
3	59	0:30
4	72	0:40
5	72	7:00
6	4	pause

Steps 2 to 4 were repeated 30x.

Picture no. 614 lower line

T7E1 assay for CCR5delta32 positive control

volume	component
5.5 µl	CCR5delta32 DNA (209 ng)
12.5 µl	H2O
2 µl	NEB10x

10.08.17

PCR for T7 assay, annealing and T7 incubation

Experiment failed.

Mycoplasm PCR

volume	volume	component
2 µl		template
1.5 µl	13.5 µl	10x Y Buffer
0.3 µl	2.7 µl	dNTPs
1 µl	9 µl	primer fw
1 µl	9 µl	primer rv
0.2 µl	1.8 µl	Taq
9 µl	81 µl	H2O

Program: myco

step	temperature (°C)	time
1	95	5:00
2	95	0:40
3	60	0:40
4	72	0:40
5	72	5:00
6	4	pause

Steps 2 to 4 were repeated 30x.

Picture no. 621

11.08.17

KO-confirm primer 228 and 229

To confirm knockout a PCR was performed.

volume	volume	component
2 µl		template
1.5 µl	7.5 µl	10x buffer
0.5 µl	2.5 µl	dNTPs
0.75 µl	3.75 µl	primer fw
0.75 µl	3.75 µl	primer rv
0.2 µl		Taq
9.3 µl	46.5 µl	H2O

PCR for T7E1 assay

PCR was performed as previously described (double volume 50 µl).

volume	component
1 µl	gDNA
10 µl	5x buffer
1.5 µl	dNTPs (10µM)
1.25 µl	primer fw
1.25 µl	primer rv
0.5 µl	phusion

Test digest HIF pcDNA3

volume	component
5 µl	DNA HIF pcDNA3
1 µl	CutSmart buffer
1 µl	XhoI
3 µl	H2O

Test digest was performed for 1 hour at 37°C (was accidentally thrown away).

Test digest of lenti-Cas9-sgRNA20 and knockdown plamsids

lenti-Cas9-sgRNA20 and knockdown SGEN112_2.1 and SGEN112_2.2 were test digested.

volume	component
1 µl	BamHI
2 µl	Plasmid
1.5 µl	FD buffer
10.5 µl	H2O

Lenti-Cas9

volume	component
1 µl	NcoI
2 µl	plasmid
1.5 µl	FD buffer
10.5 µl	H2O

T7E1 assay

50µl PCR samples (from 11.08.17) were annealed for longer than 3 h in heatblock, then 10 min at 95°C and cooled down in block. 400 ng/sample PCR product were obtained (reaction mix as previously; time: 18, 20, 22 min T7E1 at 37°C, stop with 3 µl 6x Orange Loading Dy on ice. Jurkat HIF1A exon 5 from PCR on 08.08.17 served as negative control and was incubated for 20 min with T7. 2% EtBR gel was used. Expected bands at 200 bp and 300 bp were observed in all three CCR5delta32 samples with T7.

13.08.17

KO-confirm primer 228 and 229

volume	volume	component
2 µl		template
1.5 µl	7.5	10x buffer
1 µl	5 µl	dNTPs
1 µl	5 µl	primer fw
1 µl	5 µl	primer rv
0.2 µl		Taq
8.2 µl	41 µl	H2O

program: koconfirm
picture no. 633

Retrafo of sgRNA20_4

volume	component
2 µl	template (1:1 genomic:plasmid)
1.5 µl	buffer 10x
1 µl	dNTPs
1 µl	primer fw
1 µl	primer rv
0.2 µl	Taq
8.3 µl	H2O

Program: PCR kitKOconfirm

step	temperature (°C)	time
1	94	5:00
2	94	0:40
3	60	0:40
4	72	4:00
5	72	5:00
6	4	pause

Steps 2 to 4 were repeated 35x.

14.08.17

PCR KO-Kit confirm (TAq and Q5)

A PCR was performed to confirm knockout.

volume	component
2 µl	template gDNA
1.5 µl	buffer 10x
1 µl	dNTPs
1 µl	primer fw 32
1 µl	primer rv 60
0.5 µl	Taq Q5
7.5 µl	H2O

Program: kokit3primers

step	temperature (°C)	time
1	95.0	2:00
2	95.0	0:30
3	55.5	0:40
4	72.0	2:00
5	72.0	5:00
6	4.0	pause

Steps 2 to 4 were repeated 35x.

Product ~1.6 kb at 54.5°C, ET 1.5 min
Picture no. 642

Gibson HIF1A

Insert HIF1A 20 ng/µl, 520 bp
Backbone pcDNA3 8 ng/µl, 7464 bp
Insert:backbone 4:1

volume	component
10 µl	2x gibson
9 µl	backbone
1 µl	insert

Assembly was performed for 1 h at 50°C.

XL10gold were transformed using heat shock protocol (06.05.17).

15.08.17

PCR KO-Kit confirm 3 primers

A PCR was performed to confirm knockout.

volume	component
2 µl	template gDNA
1.5 µl	buffer 10x
1 µl	dNTPs
1 µl	primer fw 32
1 µl	primer rv 60
0.3 µl	Taq (Q5)
7.7 µl	H2O

Program: kokit confirm 3 primers
at 34,5°C, elongation time 1.5 min
picture no. 648

volume	component
2 µl	template gDNA
1.5 µl	buffer 10x
1 µl	dNTPs
1 µl	primer fw 32
1 µl	primer rv 60
0.5 µl	Taq (Q5)
7.5 µl	H2O

Program: kokit confirm 3 primers
at 49°C, elongation time 2 min
Picture no. 648

Miniprep of Lenti-Cas sgRNA20_4 and test digest with EcoRI and BamHI

Miniprep was performed according to manufacturer's protocol.

volume	component
2 µl	DNA
0.75 µl	EcoRI
0.75 µl	BamHI
1.5 µl	10x FD
5 µl	H2O

PCR CaHIF1A

Protocol 13.08.17
only backbone, 1 µl dNTPs instead

16.08.17

PCR KO-Kit

gDNA101 template was diluted 1:10 diluted.

Primers 228 and 229

volume	component
1 µl	template
2 µl	buffer
1 µl	primer fw
1 µl	primer rev
1 µl	dNTPs
0.5 µl	Taq (Q5)
13.5	H2O

Primers 32 and 60

volume	component
1 µl	template
2 µl	buffer
1 µl	primer fw
1 µl	primer rev
1 µl	dNTPs
0.5 µl	Taq (Q5)
13.5	H2O

Picture no. 654

PCR on CaHIF1a gel, gelex, gibson and trafo

Performed as described previously.

Preparation of buffers for electrophoresis

Stacking gel stock: Tris-HCL 1 M pH 6.8
Separation gel stock: Tris-HCL 1.5 M pH 8.8
WB running buffer 10x: Tris 0.5 M, 0.1% SDS, glycin 1 M, HCl –> pH 8.3
10x Lämmli running buffer (1 l): 30 g tris, 10 g SDS, 144 g glycin

Protein extraction from HEK

HRE-SEAP and CoCl2 treated cells were lysed to extract proteins (12 well plate triplicates, WT, 0µM / 200 µM / 500 µM CoCl2).

Protocol cell lysis

on ice 2x washing in PBS (remove by 2 min, 100xg) with 100µM CoCl2 for CoCl2 cells
150 µl RIPA buffer, 0.1% proteinase inhibitor cocktail with 100µM CoCl2 for CoCl2 cells
5 min ultrasound
20 min 20,000xg, 4°C –> take supernatant (pellet stored for control)

SDS-PAGE

Protocol SDS-PAGE

8% acrylamid gel
4.5% stacking gel
heat samples 10 min 96°C in loading dye
1x Lämmli running buffer
run at 80 V until end of stacking gel
150 V for rest of run (until blue loading dye elutes)

Western Blot

Protocol Western Blot

assemble in running buffer
incubate/soak gel, whatman papers, sponges in running buffer
activate PVDF membrane in 100% methanol for 1 min, then wash MeOH off with running buffer
assembly: sponge, whatman paper, PVDF membrane, gel, whatman paper, sponge (every step flatten stack by rolling out from centre to outside)
run 10-12 h const. 20mAmp (also possible: 1:35 h 44 Amp/45 V)

17.07.17

Colonies picked from CaHIF1A cloning

2 colonies were picked.

18.08.17

CaHif1A miniprep, test digest and glycerol stocks

Miniprep was performed on overnight cultures CaHIF1A C1 and C2 according to manufacturer's protocol (zymo).

C1 test digest (see 11.08.17)

volume	component
1.2 µl	plasmid (745 ng/µl)
1 µl	CutSmart
1 µl	XhoI
6.8 µl	H2O

C2 test digest

volume	component
3 µl	plasmid (372 ng/µl)
1 µl	CutSmart
1 µl	XhoI
5 µl	H2O

Glycerol stocks of CaHif1A were produced.

20.08.17

KO-Kit confirm PCR

To confirm knockout a PCR was performed.

Mix 1

volume	volume	component
1 µl		template
1.5 µl	13.5 µl	buffer 10x
1 µl	9 µl	primer 228
1 µl	9 µl	primer 229
1 µl	9 µl	dNTPs
0.5 µl	4.5 µl	Taq Q5
9 µl	81 µl	H2O

Mix 2

volume	volume	component
1 µl	9 µl	template
1.5 µl	13.5 µl	buffer 10x
1 µl	9 µl	primer 60
1 µl	9 µl	primer 32
1 µl	9 µl	dNTPs
0.5 µl	4.5 µl	Taq Q5
9 µl	81 µl	H2O

Picture in lab book.

Cell lysis of JK cells

Lysis of JK cells was performed according to protocol.

21.08.17

SDS-PAGE and Western blot

SDS-PAGE were ran for ~ 2 h, Western blots overnight (const. 20 mA, 4°C).

22.08.17

Blocking Western blots

Blots were blocked for ~ 6 h, then stored at 4 °C (picture in lab book).

Generated BSA standard in TBS-T

BSA concentration	No.	lambda	Absorbance
0 µg/ml	2	620nm	0.005A
0 µg/ml	2	620nm	0.007A
0 µg/ml	2	620nm	0.007A
5 µg/ml	3	620nm	0.140A
5 µg/ml	3	620nm	0.161A
5 µg/ml	3	620nm	0.115A
10 µg/ml	3	620nm	0.275A
10 µg/ml	3	620nm	0.284A
10 µg/ml	3	620nm	0.271A
20 µg/ml	3	620nm	0.581A
15 µg/ml	3	620nm	0.435A
15 µg/ml	3	620nm	0.429A
15 µg/ml	3	620nm	0.430A
20 µg/ml	3	620nm	0.545A
20 µg/ml	3	620nm	0.574A
30 µg/ml	3	620nm	0.830A
30 µg/ml	3	620nm	0.886A
30 µg/ml	3	620nm	0.887A

24.08.17

SDS-PAGE

Protein concentration was determined using amido black stain. 10 µg protein for each sample were was loaded on gel (problem with CoCl2 treated sample: not able to load appropriate volume). Blotting occurred over night at 4 °C (20 mA, 12 h). Membrane was blocked with 5% milk powder in TBST for 2.5 h.

cell line	JK	JK	JK	JK	HEK
CoCl2 (µM)	0	200	400	800	500

26.08.17

lenti-Cas9-puro all RNAs electroporation

All RNA coding lenti-Cas9-puro were electroporated into Jurkat using CAS9-GFP RNAscramble as transfection control.
Protocol from cell culture was used:

2×10^6 cells and 9 µg DNA were resuspended in 200 µl 3P buffer
put in cuvettes and electroporated
500 µl RPMI were added
plated on 12 well plate, filled to 2 ml with RPMI

Puromycin toxicity assay

HEK cells were transfected with lenti-Cas9-puro RNA5 old cloning and SGEN as transfection control using PEI (1x10cm dish at ~50% confluency). 1 ml serum, phenol red free DMEM and 10 µg DNA were vortexed. 30 µl PEI (1µg/ml) were added and vortexed carefully. After 15 min PEI/DNA was added evenly to dish.

31.08.17

SDS-PAGE

Protein amount was determined using amido black stain. 10 µg protein for each sample were loaded. SDS-Page took ~ 2 h and blotting 2 h at 250 mA. Membranes were blocked overnight at 4°C. No results could be observed.

Cell line	HEK	HEK	HEK	JK	JK	JK	JK
CoCl2 (µM)	0	200	500	0	400	200	800

03.09.17

SDS-PAGE with new samples

Treating blots made on 31.08.17 with new ECL showed no results. Western blots were repeated but without success.

10.09.17

Cell lysis and determining protein concentration

Cell lysis was performed according to protocol (6cm dish JK with 150 µl RIPA, 6 well HEK in 250 µl RIPA). Protein concentration was determined using amido black stain.

11.09.17

SDS-PAGE and Western Blot

10 µg protein for each sample were loaded on gel for SDS-PAGE (0.02 A in stacking-gel, 0.05 A in running-gel). Blotting was performed at 250mA for 2 h. Blocking occured at room temperature for 2 h in 5% milk-TBS-T. Blots were incubated with primary antibodies overnight (anti-HIF1A, anti-GAPDH).

cell line	JK	JK	JK	JK	HEK	HEK	HEK	HEK	HEK
CoCl2 (µM)	0	200	400	800	0	200	400	600	800

12.09.17

Washing blots

Blots were washed with TBS-T 3x10min and incubated with 1:10,000 secondary antibody for 1 h (anti-mouse HRp –> HIF1A; anti-rabbit HRP –> GAPDH).

14.09.17

Lysis of JK cells and determining protein concentration

JK cells were induced with CoCl2 24 h before lysis. Lysis was performed in 150 µl RIPA and 100 µM CoCl2. Protein concentration was determined with amido black stain.

15.09.17

SDS-Gel for JK

Samples were collected one day prior to running the gel. Lysis was performed according to protocol. Amido black stain was used to determine protein amount. Proteins were blotted for 2 h at 250 mA. The mambrane was blocked for 2 h at room temperature.

cell line	ladder	JK	JK	JK	JK	JK	JK
CoCl2 (µM)		0	20	40	80	160	200

17.09.17

SDS-PAGE

Gel performed according to protocol. Western blot was performed for 2 h at 250 mA. Protein amount was determined using amido black stain. Blots were blocked for 2 h at room temperature and primary antibody was added over night.

cell line	ladder	HEK:133	HEK:133	HEK:133	HEK:133	HEK:WT	HEK:WT	HEK:WT
CoCl2 (µM)		0	200	400	600	0	400	600

cell line	ladder	HEK:134	HEK:134	HEK:134	HEK:134	HEK:WT	HEK:WT	HEK:WT
CoCl2 (µM)		0	200	400	600	0	400	600

18.09.17

SDS-PAGE and Western Blot

SDS-PAGE and Western Blot were performed according to protocol

cell line	ladder	JK:133	JK:133	JK:133	JK:133	JK:WT	JK:WT	JK:WT	JK:WT	HEK
CoCl2 (µM)		0	80	160	200	0	80	160	200	600

cell line	ladder	HEK	JK:133	JK:133	JK:133	JK:133	JK:WT	JK:WT	JK:WT	JK:WT
CoCl2 (µM)		600	0	80	160	200	0	80	160	200

20.09.17

Electroporation of 5 gRNAs into Jurkat

Electroporation of 5 gRNAs into Jurkat cells was performed according to protocol. GFP marker was used for sorting.

21.09.17

Electroporation of 5 gRNAs into Jurkat

Electroporation of 5 gRNAs into Jurkat cells was repeated. GFP marker was used for sorting.

SDS-PAGE and Western Blot

SDS-PAGE and Western Blot were performed according to protocol.

cell line	ladder	HEK:WT	HEK:WT	HEK:WT	HEK:133	HEK:133	HEK:133	HEK:133
CoCl2 (µM)		0	400	600	0	200	400	600

cell line	ladder	HEK:WT	HEK:WT	HEK:WT	HEK:134	HEK:134	HEK:134	HEK:134
CoCl2 (µM)		0	400	600	0	200	400	600

22.09.17

Sorting GFP-positive cells of pooled electroporations

GFP-positive cells of pooled electroporations were sorted. Negative control was not mock electroporated and cound not be used for gating, hence data were gated based on samples. Single cells were seeded in 96-well plates into 50 µl mycoplasm clearance medium. Microscopy was used to check for presence of cells.

23.09.17

SDS-PAGE and Western Blot

Samples were collected one day prior to SDS-PAGE gel. Protein amount was determined by amido black stain. SDS-PAGE and Western Blot were performed according to protocol (2 gels).

cell line	ladder	JK:WT	JK:WT	JK:WT	JK:WT	JK:133	JK:133	JK:133	JK:133
CoCl2 (µM)		200	80	40	0	200	80	40	0

24.09.17

Adding medium to seeded single cell plates

100 µl conditioned RPMI was added to seeded single cell plates. In the following weeks regular check of clonal cultures and addition of conditioned RPMI if necessary.

SDS-PAGE and Western Blot

Samples were collected on 23.09.17. Protein amount was determined by amido black stain. Lysis of cells, SDS-PAGE and Western Blot were performed according to protocol.

cell line	ladder	HEK:WT	HEK:WT	HEK:WT	HEK:133	HEK:133	HEK:133	HEK:133
CoCl2 (µM)		0	400	600	0	200	400	600

cell line	ladder	HEK:WT	HEK:WT	HEK:WT	HEK:134	HEK:134	HEK:134	HEK:134
CoCl2 (µM)		0	400	600	0	200	400	600

29.09.17

SDS-PAGE and Western Blot

Experiments were performed according to protocol (see day before). CMV:HIF served as HA positive control.

anti-HA

cell line	WT(HEK)	WT(HEK)	WT(HEK)	WT(HEK)	133(HEK)	133(HEK)	133(HEK)	133(HEK)
CMV:HIF	-	-	+	+	-	-	+	+
CoCl2 (µM)	0	400	0	400	0	400	0	400

cell line	WT(HEK)	WT(HEK)	WT(HEK)	WT(HEK)	134(HEK)	134(HEK)	134(HEK)	134(HEK)
CMV:HIF	-	-	+	+	-	-	+	+
CoCl2 (µM)	0	400	0	400	0	400	0	400

anti-HIF1A

cell line	WT(HEK)	WT(HEK)	WT(HEK)	WT(HEK)	133(HEK)	133(HEK)	133(HEK)	133(HEK)
CMV:HIF	-	-	+	+	-	-	+	+
CoCl2 (µM)	0	400	0	400	0	400	0	400

cell line	WT(HEK)	WT(HEK)	WT(HEK)	WT(HEK)	134(HEK)	134(HEK)	134(HEK)	134(HEK)
CMV:HIF	-	-	+	+	-	-	+	+
CoCl2 (µM)	0	400	0	400	0	400	0	400

30.09.17

Sample preparation, SDS-PAGE and Western Blot

Sample preparation, SDS-PAGE and Western Blot as described earlier.

cell line treatment	WT	WT	134	134	134	134	ladder	134 + CRE + Forskolin	134 + CRE + TDAG, pH7.5	134 + CRE + TDAG, pH7.5
CMV:HIF	-	-	-	-	+	+		-	-	-
CoCl2 (µM)	0	400	0	400	0	400		0	0	400

cell line treatment	WT	WT	134	134	134	134	ladder	134 + CRE + Forskolin	134 + CRE + TDAG, pH6.5	134 + CRE + TDAG, pH6.5
CMV:HIF	-	-	-	-	+	+		-	-	-
CoCl2 (µM)	0	400	0	400	0	400		400	0	400

→ no results

04.10.17

Sample preparation, SDS-PAGE and Western Blot

Experiments were performed as described earlier.

cell line	ladder	WT	WT	WT	WT	130	130	130	130
CoCl2 (µM)		0	200	400	600	0	200	400	600

05.10.17

Sample preparation, SDS-PAGE and Western Blot

Experiments were performed as described earlier.

cell line	WT	WT	WT	130	130	130	ladder	GFP positive
CoCl2 (µM)	0	100	200	0	100	200

cell line	ladder	WT	WT	WT	WT	130	130	130	130
CoCl2 (µM)		0	100	150	200	0	100	150	200

→ no result

06.10.17

Sample preparation, SDS-PAGE and Western Blot

Experiments were performed as described earlier.

cell line	ladder	WT	WT	WT	WT	HRE	HRE	HRE	HRE
CoCl2 (µM)		0	400	0	400	0	400	0	400
CMV:HIF		-	-	+	+	-	-	+	+

→ no results

07.10.17

T7E1 assay on puromycin selected non-clonal Jurkat HIF1A-KO

T7E1 assay was performed according to protocol. PCR for HEK CCRdelta32 positive control and WT/KOJK HIF1A exon 6 (KO1) and exon 5(KO3) were each performed twice. gDNA was extracted according to protocol in cell cluture lab book (16.05.17).

sgRNA1 vs. exon 6
sgRNA3 vs. exon 5
WT
3/4 of semi-confluent 6 cm dish for each

volume	component
x	gDNA (300-400 ng)
10 µl	5x buffer
1.5 µl	dNTPs
1.25 µl	each primer
0.5 µl	Q5
35.5-x µl	H2O

protocol HIF-KO confirmation
annealing at 59°C, 40 cycles

Very good concentrations were obtained by PCR clearup (Qiagen), no effect of template concentration on product was observed.

8.10.17

Annealing and T7 Assay

500 ng per sample in 1x NEB2, each condition double amount annealed (for +/- T7)
HEKCCR5delta32 / JK KO1 pure / JK KO1:WT ex 6 1:1 / JK WT ex 6 pure / JK KO3 pure / JK KO3:WT ex 5 1:1 / JK WT ex 5 pure
sample volume adjusted to highest sample volume
10 min 95°C in heatblock, closed with parafilm, passive cooldown in heatblock ~ 3 h

T7

12 µl annealed DNA per sample mixed on ice with 1.5 µl of 1:1 mix T7E1 and NEB2 2x buffer
20 min at 37°C in PCR cycler
stoped with 6x Orange loading dye on ice
loaded on 2% agarose EtBr gel with untreated control for each sample
run at 95 V for 50 min
positive control did not work
500 ng per sample in 1x NEB2

11.10.17

Kryo stocks Cas9-puro gRNA1 and gRNA3

Kryo stocks of puromycin selected, non clonal Jurkat cells were generated according to protocol. From 1 confluent 6 cm dish cryostock was generated by storing cells in 1 ml containing 90% FBS and 10% DMSO. After 2 days at -80°C cells were stored in liquid nitrogen tank.

13.10.17

Sample preparation, SDS-PAGE and Western Blot

Experiments were performed as described earlier.

cell line	Jurkat,WT								HEK,WT
	CoCl2[µM]	0	20	40	80	160	320	la	0	200	400

cell line	HEK, HRE								Jurkat, HRE
CoCl2[µM]	0	200	400	la	0	20	40	80	160	320

15.10.17

Transient AND-gate test

6-well plates of HEK KD134 cells were transfected using PEI to test AND-gate transiently.

PEI transfection protocol

seed cell one day before transfection, cell density should be 70-80%
PEI transfection
1. mix 3 µg DNA in 100 µl serum-free medium
2. mix 9 µg PEI in 100 µl serum-free medium
3. incubate both tubes 10 min at room temperature
4. add the PEI solution to DNA solution and mix, then incubate 15 min at RT
5. remove medium from cells
6. add 800 µl serum-free medium to DNA/PEI mixture and add to the cells
7. incubate ~3 h at 37°C
8. addd 2 ml culture medium to cells
Change medium after 3-5 h

DNA: 1 µg per plasmid, 3 µg total in each transfection
PEI: 3:1 per DNA, i.e. 9 µg per transfection

KD134

	CRE:HIF	HRE:LUC	pUC
400 µM CoCl2	pH7.7	pH7.1	pH6.1
400 µM CoCl2	pH7.7 + Forskulin + IBMX	pH7.1 + Forskulin + IBMX	pH6.1 + Forskulin + IBMX

KD134

	CRE:HIF CMV:TDAG HRE:LUC	CRE:HIF HRE:LUC SV40:TDAG	CMV:HIF HRE:LUC pUC
0 µM CoCl2	pH6.5 + Forskulin + IBMX	pH6.5 + Forskulin + IBMX
400 µM CoCl2	pH6.5 + Forskulin + IBMX	pH6.5 + Forskulin + IBMX

0 µM CoCl2	HRE:Luc 1:2 pUC
400 µM CoCl2	HRE:Luc 1:2 pUC

WT

0 µM CoCl2	HRE:Luc 1:2 pUC
400 µM CoCl2	HRE:Luc 1:2 pUC

16.10.17

Induction of transient AND-gate test

AND-gate was induced 24 h after start of PEI by regulating pH and adding CoCl2 and Forskulin according to schemes above (Forskulin 1:1000 of 100 mM stock, IBMX 1:1000 of 100 mM stock).

17.10.17

Change of all media

All media were changed 12 h after induction, all conditions were added as before to achieve more constant pH induction.

18.10.17

SDS-PAGE

SDS-PAGE was performed as previously.

cell line	WT		KD CRE:HIF, HRE:LUC, SV40:TDAG8
pH	-	6.5	7.7	7.1	6.5	7.7	7.1	6.5	-
CoCl2 [µM]	400	0	400	400	400	400	400	400	400
Ftl	-	+	+	+	+	-	-	-	-

cell line	WT		KD CRE:HIF, HRE:LUC, CMV:TDAG8
pH	-	6.5	7.7	7.1	6.5	7.7	7.1	6.5	-
CoCl2 [µM]	400	0	400	400	400	400	400	400	400
Ftl	-	+	+	+	+	-	-	-	-

20.10.17

Sample preparation, SDS-PAGE and Western Blot

Experiments were performed as described earlier.

cell line	WT				WT HRE			HIF1A shRNA2			HIF1A shRNA2 HRE
CoCl2 [µM]	0	400	0	400	0	400	0	400

cell line	WT	CRE:HIF1A,HRE:LUC						HIF1A shRNA2 CMV:HIF1A,HRE:LUC
CoCl2 [µM]	400	400	400	400	400	400	400	0	400
pH		6.5	7.1	7.7	6.5	7.1	7.7
Forskolin+IBMX		-	-	-	+	+	+

23.10.17

Sample preparation, SDS-PAGE and Western Blot

Experiments were performed as described earlier.

cell line	WT		WT CMV:HIF1A		la	WT		WT CMV:HIF1A		WT,CMV:HIF1A_2
CoCl2 [µM]	0	400	0	400		0	400	0	400	400

cell line	la	JK HRE, high
CoCl2 [µM]		0	5	10	20	40	80	160	320

Team:Freiburg/Notebook Knockout

Lab Notebook Knockout

06.05.2017

Retransformation of lenti-CAS9-puro plasmid

07.05.2017

Producing overnight cultures

Test digest of lenti-CAS9-puro

08.05.2017

Miniprep of lenti-CAS9-puro

09.05.2017

Test digest of lenti-CAS9-puro

10.5.2017

Test digestion of Cathomen Cas9 plasmid

11.05.2017

Retransformation of knockdown plasmids

12.05.2017

Producing overnight cultures of SDEN, SBEN, EDEN

13.05.2017

Retransformation of CAS9-GFP plasmid

Miniprep of SDEN, SBEN, EDEN

14.05.2017

Producing overnight cultures CAS9-GFP

15.05.2017

Miniprep of CAS9-GFP

Testdigest of CAS9-GFP

18.05.2017

PCR for HIF1A exons

19.05.2017

Gel extraction

Retransformation of KO-Kit plasmids

20.05.2017

Miniprep and Midiprep of several plasmids

23.05.2017

Digestion of CAS9-GFP by BbsI

Annealing of sgRNA oligos

Testdigest of KO-Kit plasmids

Midiprep of SBEN, lenti-CAS9-puro

25.05.17

Testdigest of KO-KIT (101,104,203) with EcoRV

Ligation of sgRNAs 1;3 into CAS9 GFP

26.05.17

Sitedirected mutagenesis (SDM) Q5 PCR with sgRNA overhang primers.

Ligation of sgRNA in CAS9-GFP

27.05.17

Test of Midi Prep Kit

28.05.17

SDM PCR sgRNA1 CAS9GFP gradient

29.05.17

SDM PCR for all sgRNAs CAS9-GFP

30.05.17

BsmBI digest of lenti-CAS9-puro (AG Cathomen)

SDM PCR of sgRNA1 vs. ctrl

31.05.17

SDM PCR for sgRNA5;20

Gelextraction of sgRNA1 in CAS9-GFP

BsmBI digest of lenti-CAS9-puro

01.06.17

CAS9-GFP-sgRNA1 T4 ligation & DpnI digest

Maxi Prep Kit of KO-KIT plasmids

02.06.17

SMD PCR CAS9-GFP sgRNA 1;5;20;ctrl

BsmBI digest of lenti-CAS9-puro

SDM PCR CAS9-GFP-sgRNA5

Inoculation of 5ml culture LB-Amp with XL10gold

03.06.17

BsmBI digest of lenti-CAS9-puro

Midi Prep

SDM PCR of CAS9-GFP sgRNA5

04.06.17

PCR eGFP-fragment for SGEN plasmid

PCR SDEN short fragment for SGEN plasmid

SDEN BamHI digest

CAS9-GFP-sgRNA 5 Ligation

Oligocloning sgRNA1/3 in lenti-CAS9-puromycin

Transformation of XL10gold via heatshock transformation

05.06.17

Digest of lenti-CAS9-puromycin

SDM PCR for CAS9-GFP-shRNA3;4;9

06.06.17

Miniprep of overnight lenti-CAS9-sgRNA3