All protocols of our work can be found on Protocols page.

PCR primers sequences can be found here.

21.06.15

In the lab today: Shai and Shoham

• We received from Addgene 2 plasmids (in bacterial stab): dCas9-vp64 (#47107) and SaCas9 (#61591). they were spread on LB agar plates with ampicillin ON at 37°C.
• We transformed pSB1C3 backbone into DH5α E. coli and seeded it on LB agar plates with Chloramphenicol.

22.06.15

In the lab today: Shai and Shoham

• 10ml of LB (+ampicillin) was inoculated with one colony from dCas9-vp64 LB agar plate.
• The same was done for SaCas9.

28.06.15

In the lab today: Shai and Shoham

• 10ml of LB (+ampicillin) was inoculated with one colony from dCas9-vp64 LB agar plate.
• The same was done for SaCas9.

29.06.15

In the lab today: Shai

• 10ml of LB (+Chloramphenicol) was inoculated with one colony from pSB1C3 LB agar plate.
• We performed miniprep for dCas9-vp64 and SaCas9.

30.06.15

In the lab today: Shai and Shoham

• SaCas9 (DH5α) glycerol stock was prepared.
• dCas9-vp64 (DH5α) glycerol stock was prepared
• We performed miniprep for pSB1C3.

01.07.15

In the lab today: Shai and Shoham

• Restriction of pSB1C3 backbone with EcoRI and PstI. Alkaline phosphatase was added. Incubation for 30 minutes at 37°C.

We prepared 1% Agarose gel, and run the restriction products.

We extracted the appropriate DNA band (according to size) and performed gel extraction protocol. Nanodrop concentration results were 60 ng/μl.

• Received uMLP,gMLP,uUBB,gUBB (table for abbreviation is needed) from synthesis in a form of DNA fragments.

Restriction of uMLP,gMLP,uUBB,gUBB with EcoRI and PstI. Incubation for 45 minutes at 37°C.

The restriction products were purified using PCR purification kit. Nanodrop concentration results were 3ng/μl.

• Ligation of pSB1C3 (vector) with uMLP,gMLP,uUBB,gUBB (insert). (each time with different insert).
• 2X10ml of LB (+Chloramphenicol) was inoculated with one colony from pSB1C3 LB agar plate.

06.07.15

In the lab today: Shoham

• Ligation products from 01.07 (pSB1C3+uMLP,gMLP,uUBB,gUBB) were transformed into DH5α and seeded on LB agar plates with Chloramphenicol ON.
• Received virus kit from Agilent (cat. No. 240071) (the kit includes helper plasmids, RC plasmids, AAV-MCS plasmids, HEK cells for virus production).
• We transformed pAAV-MCS into DH5α and seeded it on LB agar plates with ampicillin.

07.07.15

In the lab today: Shoham

• No colonies found on any of the plates of 01.07 ligations.
• Colonies found on pAAV-MCS plates from yesterday. 10ml of LB (+ampicillin) was inoculated with one colony.

08.07.15

In the lab today: Shoham

• We performed miniprep for pAAV-MCS. Nanodrop concentration results were 110ng/μl and 117ng/μl (2 Eppendorfs).
• PCR was carried out on gMLP, gUBB, uMLP, uUBB using default PCR program.

The PCR products were purified using PCR purification kit.

Nanodrop concentration results:

gMLP : 96.6ng/μl, gUBB: 40.5ng/μl, uMLP: 38.0ng/μl, uUBB: 26.0ng/μl.

We prepared 1% Agarose gel, and run the PCR products to check whether the PCR worked as expected. Gel results were good (band in expected size).

Restriction of PCR products with EcoRI and PstI. Incubation for 35 minutes at 37°C.

The restriction products were purified using PCR purification kit. Nanodrop concentration results:

gMLP : 18.8ng/μl, gUBB: 6.1ng/μl, uMLP: 6.7ng/μl, uUBB: 9.4ng/μl.

12.07.15

In the lab today: Shai and Shoham

• Restriction of pSB1C3 backbone with EcoRI and PstI. Alkaline phosphatase was added. Incubation for 30 minutes at 37°C.

We prepared 1% Agarose gel, and run the restriction products.

We extracted the appropriate DNA band (according to size) and performed gel extraction protocol.

• Ligation of pSB1C3 (vector) with uMLP from 08.07(insert).
• Ligation of pSB1C3 (vector) with uUBB from 08.07 (insert).
• Ligation products (pSB1C3+uMLP, pSB1C3+uUBB) were transformed into DH5α and seeded on LB agar plates with Chloramphenicol ON.
• We received from synthesis by Syntezza Bioscience Ltd. hTERT promoter (in pU257 plasmid). It was spread on LB agar plates with ampicillin ON at 37°C.

13.07.15

In the lab today: Shai and Shoham

• PCR was carried out again for gMLP, gUBB, uMLP, uUBB using default PCR program.

The PCR products were purified using PCR purification kit.

Nanodrop concentration results:

gMLP : 106.7ng/μl, gUBB: 249.8ng/μl, uMLP: 110.1ng/μl, uUBB: 57.6ng/μl.

Since concentrations of gMLP, uMLP, uUBB are low we tried to run them in purification columns again with 10μl of elution buffer.

New nanodrop concentration results were:

gMLP : 97.2ng/μl, uMLP: 47.4ng/μl, uUBB: 73.1ng/μl.

• 10ml of LB (+ampicillin) was inoculated with one colony of pAAV-MCS plate from 07.07.

14.07.15

In the lab today: Shai, Shoham and Vlad

• Shoham - Cells expermints - add later
• We prepared 1% Agarose gel, and run the PCR products from yesterday to check whether the PCR worked as expected. Gel results indicate PCR was fine.



• We performed miniprep for pAAV-MCS from yesterday. Nanodrop concentration results were 169.2ng/μl.
• Colonies found on hTERT plates from 12.07. 10ml of LB (+ampicillin) was inoculated with one colony.

15.07.15

In the lab today: Shai, Shoham, Vlad and Bar

• Restriction of PCR products from 13.07 (gMLP, gUBB, uMLP, uUBB) with EcoRI and PstI. Incubation for 35 minutes at 37°C.

We prepared 1% Agarose gel, and run the restriction products of gUBB and uUBB.

We extracted the appropriate DNA band (according to size) and performed gel extraction protocol. Because of human error, uUBB extraction failed. Nanodrop concentration of gUBB: 26.1ng/μl (1.83/2.38).

gMLP and uMLP restriction products were purified using PCR purification kit (so we can compare if one of the purification methods gives better results than the other). Nanodrop concentration results:

gMLP : 31.7ng/μl (1.75/1.55), uMLP: 12.5ng/μl (2.24/1.01).

• We performed miniprep for hTERT from yesterday. Then restrictions with EcoRI and PstI. Restrictions done in incubation for 30 minutes at 37°C.

We run restriction products on the gel prepared earlier today, extracted the appropriate DNA band (according to size) and performed gel extraction protocol.

16.07.15

In the lab today: Shai, Shoham

• Restriction of pSB1C3 backbone with EcoRI and PstI. Alkaline phosphatase was added. Incubation for 30 minutes at 37°C.

We prepared 1% Agarose gel, and run the restriction products.

We extracted the appropriate DNA band (according to size) and performed gel extraction protocol.

• Ligation of pSB1C3 (vector) with hTERT from yesterday (insert).

Ligation products (pSB1C3+hTERT) were transformed into DH5α and seeded on LB agar plates with Chloramphenicol ON.

• Ligation of pSB1C3 (vector) with gMLP from yesterday (insert).
• Ligation of pSB1C3 (vector) with uMLP from yesterday (insert).
• PCR was carried out on ligation products (pSB1C3+gMLP, pSB1C3+uMLP) to check whether the ligation worked as expected (using F primer of pSB1C3 and R primer of insert). We prepared 1% Agarose gel, and run the PCR products. Gel results were good (band in expected size) so ligation products were transformed into DH5α and seeded on LB agar plates with Chloramphenicol ON.

GEL IMAGE!!!

17.07.15

In the lab today: Shoham

• No colonies found on any of the plates from yesterday.

18.07.15

In the lab today: Shai

• 10ml of LB (+Chloramphenicol) was inoculated with one colony from pSB1C3 LB agar plate.

19.07.15

In the lab today: Shai, Shoham

• Ligation products of pSB1C3+gMLP, pSB1C3+uMLP from 16.07 were transformed again into DH5 α and seeded on LB agar plates with Chloramphenicol ON.
• We performed miniprep for pSB1C3 from yesterday. Nanodrop concentration results were 85.2ng/μl (2.04,1.84).
• PCR was carried out on gMLP, gUBB, uMLP, uUBB using default PCR program (except Tm was changed form 60°C to 65°C).

The PCR products were purified using PCR purification kit.

Nanodrop concentration results:

gMLP : 100.1ng/μl (1.79,0.72), uMLP: 139.8ng/μl (1.81,1.78),

gUBB : 81.1ng/μl (1.80,1.08), uUBB: 78.9ng/μl (1.83,0.60).

20.07.15

In the lab today: Shai, Shoham, Bar, Vlad

• Colonies found on pSB1C3+gMLP, pSB1C3+uMLP plates from yesterday.

Colony PCR was carried out on 2 colonies of pSB1C3+gMLP and 3 colonies of pSB1C3+uMLP to check whether the ligation worked as expected (using F primer of pSB1C3 and R primer of insert). We prepared 1% Agarose gel, and run the PCR products. Gel results were good (band in expected size).

3X10ml of LB (+Chloramphenicol) was inoculated with 1 colony each of pSB1C3+uMLP and the same was done for the two colonies of pSB1C3+ gMLP.

• Restriction of hTERT with EcoRI and PstI. Restriction done in incubation for 30 minutes at 37°C. Restriction products ran on gel made earlier today.

We extracted the appropriate DNA band (according to size) and performed gel extraction protocol. Nanodrop concentration results were 6.9ng/μl.

GEL IMAGE!!!

21.07.15

In the lab today: Shai, Shoham, Vlad, Shalev

• We performed miniprep for pSB1C3+gMLP, pSB1C3+uMLP from yesterday. Nanodrop concentration results were:

pSB1C3 +gMLP (colony no. 1) 123.4ng/μl.

pSB1C3 +gMLP (colony no. 2) 117.0ng/μl.

pSB1C3 +uMLP (colony no. 1) 103.0ng/μl.

pSB1C3 +uMLP (colony no. 2) 95.0ng/μl.

pSB1C3 +uMLP (colony no. 3) 110.0ng/μl.

• Restrictions of pSB1C3+gMLP and pSB1C3+uMLP with XhoI (to check ligation). All restrictions done in incubation for 30 minutes at 37°C. Restriction products ran on 1% Agarose gel.

Gel results indicate ligation of pSB1C3+gMLP and pSB1C3+uMLP succeeded à

Send for sequencing.

GEL IMAGE!!!

• 10ml of LB (+Chloramphenicol) was inoculated with one colony from pSB1C3 LB agar plate.
• 10ml of LB (+ampicillin) was inoculated with one colony from hTERT LB agar plate.

22.07.15

In the lab today: Shai, Shoham, Vlad, Shalev

• Restrictions of pSB1C3 and hTERT from yesterday with EcoRI and PstI. Restrictions done in incubation for 30 minutes at 37°C. Alkaline phosphatase was added to pSB1C3.

We prepared 1% Agarose gel, and run the restriction products.

We extracted the appropriate DNA band (according to size) and performed gel extraction protocol.

Ligation of pSB1C3 (vector) with hTERT from (insert).

• Ligation of pSB1C3 (vector) with uUBB from 19.07(insert).
• Ligation of pSB1C3 (vector) with gUBB from 19.07 (insert).
• Ligation products (pSB1C3+hTERT,pSB1C3+uUBB, pSB1C3+gUBB) were transformed into DH5 α and seeded on LB agar plates with Chloramphenicol ON.

23.07.15

In the lab today: Shai and Shoham

• Colonies found on all plates from yesterday (pSB1C3+hTERT,pSB1C3+uUBB, pSB1C3+gUBB). 4 colonies were taken from each plate and 12X10ml of LB (+Chloramphenicol) was inoculated with one colony each.

24.07.15

In the lab today: Shai and Shoham

• We performed miniprep for all pSB1C3+hTERT,pSB1C3+uUBB, pSB1C3+gUBB from yesterday. Nanodrop concentration checked for all:

	pSB1C3 +hTERT (ng/μl)	pSB1C3 +uUBB (ng/μl)	pSB1C3 +gUBB (ng/μl)
LB no. 1	233	-	231
LB no. 2	309	249	154
LB no. 3	199	227	123
LB no. 4	248	246	187

• Restrictions of miniprep products with EcoRI and PstI (to check ligation). All restrictions done in incubation for 30 minutes at 37°C. Restriction products ran on 1% Agarose gel.

Gel results indicate ligation of pSB1C3+hTERT,pSB1C3+uUBB, pSB1C3+gUBB succeeded à Send for sequencing of 2 colonies from each.

GEL IMAGE!!!

26.07.15

In the lab today: Shoham

• 10ml of LB (+ampicillin) was inoculated with one colony from dCas9-vp64 LB agar plate.
• The same was done for SaCas9.

27.07.15

In the lab today: Shai and Shoham

• We performed miniprep for SaCas9, dCas9-vp64 from yesterday (2 Eppendorfs each). Nanodrop concentration results were:

	SaCas9	dCas9-vp64
Eppendorf no. 1	277ng/μl (2.01/2.06)	593ng/μl (1.85/2.09)
Eppendorf no. 2	215ng/μl (1.98/2.09)	522ng/μl (1.89/2.30)

• We received Survivin mCherry from outsourcing synthesis (which brand?).
• We transformed plasmids from virus kit (pAAV-MCS, pRC, pHelper) and Survivin mCherry into DH5α and seeded them on LB agar plates with ampicillin.

28.07.15

In the lab today: Shai and Shoham

• Colonies found on all plates from yesterday. 2 colonies were taken from each plate and 8X10ml of LB (+ampicillin) was inoculated with one colony each.

29.07.15

In the lab today: Vlad and Shalev

• We performed miniprep for pAAV-MCS, pRC, pHelper, Survivin mCherry from yesterday (2 Eppendorfs each). Nanodrop concentration results were:

	colony no. 1	colony no. 2
pHelper	602ng/μl	236ng/μl
pRC	520ng/μl	399ng/μl
pAAV-MCS	186ng/μl	137ng/μl
Survivin mCherry	305ng/μl	357ng/μl

02.08.15

In the lab today: Shai and Shoham

• We transformed pSB1C3+gMLP (from colony no. 2 at 21.07) into DH5α E. coli and inserted into 10ml of LB (+Chloramphenicol) ON.
• We transformed pSB1C3+uMLP (from colony no. 3 at 21.07) into DH5α E. coli and seeded it on LB agar plates with Chloramphenicol.
• 10ml of LB (+Chloramphenicol) was inoculated with colony no. 3 of pSB1C3+hTERT from 23.07.
• 10ml of LB (+Chloramphenicol) was inoculated with colony no. 2 of pSB1C3+uUBB from 23.07.
• We transformed pGFPN1 (GFP plasmid found on our lab) into DH5α and seeded it on LB agar plates with ampicillin.

03.08.15

In the lab today: Shai and Shoham

• Nothing grew in LB of pSB1C3+gMLP and pSB1C3+uMLP from yesterday, so we did transformation again, this time with heat shock:

pSB1C3 +gMLP (colony no. 2 from 21.07), pSB1C3+uMLP (colony no. 2 from 21.07) and pSB1C3+uMLP (colony no. 3 from 21.07) all were inserted into 10ml of LB (+Chloramphenicol) ON.

• Colonies found on pGFPN1 plate from yesterday. 10ml of LB (+ampicillin) was inoculated with one colony.

04.08.15

In the lab today: Vlad

• We performed miniprep for: pSB1C3+gMLP (colony no. 2), pSB1C3+uMLP (colony no. 2), pSB1C3+uMLP (colony no. 3), pGFPN1 (all from yesterday) and pSB1C3+hTERT from 02.08. Nanodrop concentration results were:

pSB1C3 +gMLP(2): 245ng/μl, pSB1C3+uMLP(2): 221ng/μl,

pSB1C3 +uMLP(3): 256ng/μl, pSB1C3+hTERT(3): 58.6ng/μl, pGFPN1: 2.4ng/μl.

• Restriction of pAAV-MCS and pGFPN1 with EcoRI and XbaI. Alkaline phosphatase was added to pAAV-MCS. Incubation for 30 minutes at 37°C.

We prepared 1% Agarose gel, and run the restriction products.

Gel results indicate that restriction of pGFPN1 failed.

05.08.15

In the lab today: Shai

• We performed miniprep for: pSB1C3+uUBB (colony no. 2) from 02.08. Nanodrop concentration results were 160ng/μl.
• Send for sequencing miniprep results of yesterday and today (pSB1C3+hTERT(3), pSB1C3+gMLP(2), pSB1C3+uMLP (2), pSB1C3+uMLP(3), pSB1C3+uUBB(2)).

Update from 09.08: Sequencing failed to determine DNA sequences.

06.08.15

In the lab today: Shai, Vlad

• We decided to try another GFP plasmid (instead of pGFPN1). New plasmid: pAM-EGFP.
• Restriction of pAAV-MCS and pAM-EGFP with HindIII and BamHI. Alkaline phosphatase was added to pAAV. Incubation for 30 minutes at 37°C.

We prepared 1% Agarose gel, and run the restriction products.

We extracted the appropriate DNA bands (according to size) and performed gel extraction protocol. Nanodrop concentration results were

GFP : 19.6ng/μl and pAAV: 9.7ng/μl.

GEL IMAGE!!!

• Ligation of pAAV (vector) with GFP (insert).

Ligation products (pAAV+GFP) were transformed into DH5α and seeded on LB agar plates with ampicillin ON.

09.08.15

In the lab today: Shai

• No colonies found on pAAV+GFP plates from yesterday, so we did restriction again.

Restriction of pAAV-MCS and pAM-EGFP with HindIII and BamHI. Alkaline phosphatase was added to pAAV. Incubation for 30 minutes at 37°C.

We prepared 1% Agarose gel, and run the restriction products.

We extracted the appropriate DNA bands (according to size) and performed gel extraction protocol.

• Ligation of pAAV (vector) with GFP (insert).

Ligation products (pAAV+GFP) were transformed into DH5α and seeded on LB agar plates with ampicillin ON.

• 2X100ml of LB (+ampicillin) were made for midiprep tomorrow. One was inoculated with a colony of pHelper and the other with pRC.

10.08.15

In the lab today: Shai , Shoham

• 2 colonies found on pAAV+GFP plates from yesterday. 2X10ml of LB (+ampicillin) was inoculated with one colony each.
• We performed midiprep for pHelper, pRC, pAAV-MCS. Nanodrop concentration results were:

	Vessel no. 1	Vessel no. 2
pRC	600ng/μl (1.82/2.22)	646ng/μl (1.84/2.13)
pHelper	658ng/μl (1.87/2.24)	581ng/μl
pAAV-MCS	217ng/μl (1.80/1.53)	277ng/μl (1.79/1.56)

11.08.15

In the lab today: Vlad , Shalev

• Vlad - Cells expermints - add later
• We received Master plasmid (pMaster) from IDT. It was transformed into DH5α and inserted into 10ml of LB (+ampicillin) ON.
• We performed miniprep for pAAV+GFP from yesterday.

Nanodrop concentration results were:

colony no. 1: 189.6ng/μl (1.96,2.05), colony no. 2: 182.6ng/μl (1.94,2.03).

• Restriction of pAAV+GFP with HindIII and BamHI (to check ligation). All restrictions done in incubation for 30 minutes at 37°C. Restriction products ran on 1% Agarose gel. (why restriction with the same enzymes as original restriction, and when the there is no pieces it's the good ligation?)

Gel results indicate ligation of pAAV+GFP in colony no. 2 succeeded.

GEL IMAGE!!!

12.08.15

In the lab today: Shoham, Vlad and Shalev

• pMaster (DH5α) glycerol stock was prepared
• GFP-AAV (DH5α) glycerol stock was prepared
• PCR was carried out on uMLP, uUBB, AF using default PCR program.

The PCR products were purified using PCR purification kit.

Nanodrop concentration results of uMLP were 160.2ng/μl (1.84/1.92).

Nanodrop concentration results of uUBB were 219.8ng/μl (1.85/1.08).

Nanodrop concentration results of AF were 109.0ng/μl (1.84/2.11).

We prepared 1% Agarose gel, and run the PCR products.

GEL IMAGE!!!

• We performed midiprep for pMaster. Nanodrop concentration results were 286.8ng/μl (1.87/2.28).
• Restriction of uMLP, uUBB with SalI and PacI. Incubation for 30 minutes at 37°C.

13.08.15

In the lab today: Shoham, Vlad, Bar and Shalev

• Restriction of DNA . All restrictions done in incubation for 30 minutes at 37°C. Then we run restriction products on 1% Agarose gel, extracted the appropriate DNA band (according to size) and performed gel extraction protocol. Nanodrop concentration checked.

	Restriction enzymes	Alkaline phosphatase	Nanodrop concentration (ng/μl)
pAAV_MCS	EcoRI, XhoI	Added	50
pMaster (1)	SalI, PacI	Added	56
pMaster (2)	SalI, XbaI	Added	74
pMaster (3)	XhoI, BamHI	Added	64
pSurvivin mCherry	SalI, PacI		13.2
dCas9-vp64	SalI, XbaI		43
SaCas9	XhoI, BamHI		29
AF *	EcoRI, XhoI		50

*AF was purified using PCR purification kit (not gel extraction).

• Ligations :
• Ligation of pAAV (vector) with AF (insert).
• Ligation of pMaster (vector) with uMLP (insert).
• Ligation of pMaster (vector) with uUBB (insert)
• Ligation of pMaster (vector) with dCas9-vp64 (insert).
• Ligation of pMaster (vector) with SaCas9 (insert).

After ligation, products were transformed into DH5α and seeded on LB agar plates with ampicillin ON.

15.08.15

In the lab today: Shoham

• No colonies found on any of the plates from yesterday. So we do all restrictions and ligation again.
• Restriction of DNA . All restrictions done in incubation for 30 minutes at 37°C. Then we run restriction products on 1% Agarose gel, extracted the appropriate DNA band (according to size) and performed gel extraction protocol. Since it is Saturday nanodrop room is closed so we didn't check the concentration and didn't continue to ligation (nanodrop results added later).

	Restriction enzymes	Alkaline phosphatase	Nanodrop results - eppendorf 1	Nanodrop results - eppendorf 2
pAAV_MCS (from stock)	EcoRI, XhoI	Added	-	-
pMaster (1)	SalI, PacI	Added	78.7 (2.47/0.55)	119.0(1.87/0.69)
pMaster (2)	SalI, XbaI	Added	74.5 (2.51/0.51)	68.0 (1.85/1.43)
pMaster (3)	XhoI, BamHI	Added	72.9 (2.19/0.29)	56.9 (1.94/1.04)
pSurvivin mCherry	SalI, PacI		34.2 (2.37/0.06)	11.1 (1.79/0.13)
dCas9-vp64	SalI, XbaI		43.3 (2.78/0.18)	30.3 (2.06/1.41)
SaCas9 (from stock)	XhoI, BamHI		30.0	20.0
uUBB	SalI, PacI		25.6 (5.26/0.21)	14.0 (1.67/0.30)
uMLP	SalI, PacI		21.7 (4.59/0.06)	12.8 (1.71/0.08)
AF *	EcoRI, XhoI		17.0	-

*AF was purified using PCR purification kit (not gel extraction).

GEL IMAGE!!!

• Since we can't do ligation with current products of restriction, we tried ligation of yesterday's product again:
• Ligation of pAAV (vector) with AF (insert).
• Ligation of pMaster (vector) with uMLP (insert).
• Ligation of pMaster (vector) with uUBB (insert)
• Ligation of pMaster (vector) with dCas9-vp64 (insert).
• Ligation of pMaster (vector) with SaCas9 (insert).
• Ligation of pMaster (vector) with Survivin mCherry (insert).

After ligation, products were transformed into DH5α and seeded on LB agar plates with ampicillin ON.

16.08.15

In the lab today: Shoham, Vlad

• Colonies found on pMaster + Survivin mCherry plates from yesterday. 10ml of LB (+ampicillin) was inoculated with one colony.
• Colonies found on pMaster + dCas9-vp64 plates from yesterday. 2X10ml of LB (+ampicillin) was inoculated with one colony each.
• Colonies found on pAAV + AF plates from yesterday. 10ml of LB (+ampicillin) was inoculated with one colony.
• No colonies found on the other plates from yesterday. So we do ligation with yesterday's restriction products:
• Ligation of pMaster (vector) with uMLP (insert).
• Ligation of pMaster (vector) with uUBB (insert)
• Ligation of pMaster (vector) with SaCas9 (insert).

17.08.15

In the lab today: Vlad, Shalev

• Nothing grew in LB of pMaster+Survivin mCherry from yesterday.
• LB of pMaster+dCas9-vp64 and LB of pAAV+AF are OK. Miniprep was performed on both. Nanodrop concentration results were 193.5ng/μl (1.95/20.9) for pMaster+ dCas9-vp64 and 209.7ng/μl (1.97/2.09) for pAAV+AF.

We prepared 1% Agarose gel, and run Miniprep products.

GEL IMAGE!!!

Gel results indicate that ligation of pMaster+ dCas9-vp64 failed.

Ligation of pAAV+AF succeeded.

pAAV +AF (DH5α) glycerol stock was prepared.

• Yesterday's ligation products (pMaster+uMLP, pMaster+uUBB, pMaster+SaCas9) were transformed into DH5α and seeded on LB agar plates with ampicillin ON.
• Ligation products of pMaster+dCas9-vp64 (from 15.08) were transformed again into DH5α and seeded on LB agar plates with ampicillin ON.

18.08.15

In the lab today: Shoham, Vlad, Shalev

• Restrictions again of pMaster, Survivin mCherry, uUBB, uMLP with SalI, PacI. All restrictions done in incubation for 30 minutes at 37°C. Alkaline phosphatase was added to pMaster.
• Restriction products of pMaster, Survivin mCherry ran on 1% Agarose gel.

GEL IMAGE!!!

Gel results indicate about a problem. Tomorrow we will use new pMaster from midiprep and new Survivin mCherry from glycerol stock (10ml of LB (+ampicillin) was inoculated).

• The restriction products of uUBB, uMLP were purified using PCR purification kit. Nanodrop concentration results were 21.1ng/μl for uUBB and 35.9ng/μl for uMLP.
• We inoculated 10ml of LB (+ampicillin) with one colony from pMaster+SaCas9 from yesterday.
• We inoculated 10ml of LB (+ampicillin) with one colony from pMaster+Survivin mCherry from 16.08.

19.08.15

In the lab today: Vlad, Shalev

• Nothing grew in LB of pMaster+Survivin mCherry from yesterday.
• LB of pMaster+SaCas9 is OK. Miniprep was performed.

We prepared 1% Agarose gel, and ran Miniprep products.

Gel results indicate that ligation of pMaster+SaCas9 failed.

• We performed midiprep for pMaster and miniprep for Survivin mCherry. Then restrictions of both with SalI, PacI. All restrictions done in incubation for 30 minutes at 37°C. Alkaline phosphatase was added to pMaster.

We run restriction products on the gel prepared earlier today, extracted the appropriate DNA band (according to size) and performed gel extraction protocol.

GEL IMAGE!!!

• Ligations :
• Ligation of pMaster (vector) with Survivin mCherry (insert). (Survivin from today).

Ligation of pMaster (vector) with Survivin mCherry (insert). (Survivin from 16.08).

Ligation of pMaster (vector) with Survivin mCherry (insert). (Survivin from 13.08).

Ligation of pMaster (vector) with uMLP (insert). (uMLP from 18.08).

Ligation of pMaster (vector) with uUBB (insert). (uUBB from 18.08).

After ligation, products were transformed into DH5α and seeded on LB agar plates with ampicillin ON.

20.08.15

In the lab today: Vlad, Shalev

• No colonies found on any of the plates from yesterday.
• We think maybe there is a problem with the pMaster. So we prepared 1% Agarose gel, and run ligation products from different days.

GEL IMAGE!!!

We decided to try pMaster from 2 dates: 19.08 and 16.08 for today's ligations:

Ligation of pMaster (vector) with Survivin mCherry (insert). (pMaster from 19.08).

Ligation of pMaster (vector) with Survivin mCherry (insert). (pMaster from 16.08).

Ligation of pMaster (vector) with uUBB (insert). (pMaster from 19.08).

Ligation of pMaster (vector) with uMLP (insert). (pMaster from 16.08).

After ligation, products were transformed into DH5α and seeded on LB agar plates with ampicillin ON.

21.08.15

In the lab today: Shoham, Vlad, Shalev

• No colonies found on any of the plates from yesterday.
• We start working on pRC and pHelper of AAV kit.

Both were transformed into DH5α and inserted into 10ml of LB (+ampicillin) ON.

22.08.15

In the lab today: Shoham

• We performed midiprep for pRC. Nanodrop concentration results were 3745ng/μl (1.81/2.24).
• We performed midiprep for pHelper. Nanodrop concentration results were 4072ng/μl (1.79/2.17).

23.08.15

In the lab today: Vlad, Shalev

• Since no colonies found on any of the plates in 21.08, we do DNA restrictions again.

Restriction of pMaster, Survivin mCherry with SalI and PacI. Incubation for 30 minutes at 37°C. Alkaline phosphatase was added to pMaster.

We ran restriction products on 1% Agarose gel.

GEL IMAGE!!!

Gel result indicates that restriction didn't work as expected. Maybe PacI enzyme doesn't work as expected - enzyme was replaced and restriction done again. Restriction products were separated on 1% Agarose gel.

GEL IMAGE!!!

• Gel result indicates that pMaster restriction didn't work as expected. Maybe pMaster eppendorf is contaminated. So pMaster from original stock was transformed into DH5α and inserted into 10ml of LB (+ampicillin) ON.
• Survivin mCherry gel results are OK.
• Meanwhile, we decided to try ligation with current pMaster. So both pMaster and Survivin mCherry extracted from gel.
• Since it didn't work so far, we tried to improvise with the ligation protocol. Instead of 50ng of vector, we took 100ng. Instead of 150ng of insert, we took 400ng. So the vector/insert ratio was changed from 1:3 to 1:4.

Ligation of pMaster (vector) with dCas9-vp64 (insert).

Ligation of pMaster (vector) with SaCas9 (insert).

• After ligation, products were transformed into DH5α and seeded on LB agar plates with ampicillin ON. We also made a change in transformation protocol - instead of 10 minutes in ice, we left the DH5α for 40 minutes in the ice.

24.08.15

In the lab today: Shoham, Vlad, Shalev

• We performed miniprep for pMaster from yesterday (from stock). Nanodrop concentration result was 233ng/μl.

Restriction of pMaster was made with SalI, PacI. Restriction done in incubation for 35 minutes at 37°C. Alkaline phosphatase was added.

Restriction products ran on 1% Agarose gel. pMaster gel results are good.

GEL IMAGE!!!

• Colonies found on pMaster+SaCas9 plate from yesterday. 10ml of LB (+ampicillin) was inoculated with one colony ON
• Ligations:

2X Ligation of today's pMaster (vector) with yesterday's Survivin mCherry (insert).

Ligation of yesterday's pMaster (vector) with uMLP (insert).

After ligation, products were transformed into DH5α and seeded on LB agar plates with ampicillin ON. Like yesterday, for one of pMaster+ Survivin mCherry ligations we changed the protocol - instead of 10 minutes in ice, we left the DH5α for 40 minutes in the ice.

• AAV plasmids arrived from cloning: Htert+SaCas9, Htert+dCas9-vp64, MLPE, SMLP, SUBB.

Plasmids were transformed into DH5α and inserted into 100ml of LB (+ampicillin) ON, for midiprep tomorrow.

25.08.15

In the lab today: Vlad, Shalev

• We performed midiprep for SMLP, SUBB, Htert+dCas9-vp64 . Nanodrop concentration results were:

SMLP : 1379ng/μl (1.85/2.25) - concentration is good.

SUBB : 270ng/μl (1.86/2.24) - concentration is too low.

Htert+dCas9-vp64: concentration is too low.

• Restrictions of pMaster+SaCas9 with NotI, AgeI (to check ligation). All restrictions done in incubation for 30 minutes at 37°C. Restriction products ran on 1% Agarose gel. Gel results indicate ligation of pMaster+SaCas9 failed.

GEL IMAGE!!!

• Colonies found on all plates from yesterday (2XpMaster+Survivin mCherry, 1XpMaster+uMLP). 10ml of LB (+ampicillin) was inoculated with 2 colonies each.
• Colonies found on pMaster+SaCas9 plates from 23.08. 10ml of LB (+ampicillin) was inoculated with one colony.

26.08.15

In the lab today: Vlad, Shalev

• Restrictions of pMaster+Survivin mCherry with SalI, AgeI (to check ligation). All restrictions done in incubation for 30 minutes at 37°C. Restriction products ran on 1% Agarose gel.

Gel results indicate ligation of pMaster+Survivin mCherry succeeded.

• Restrictions of pMaster+uMLP with SalI, PstI (to check ligation). All restrictions done in incubation for 30 minutes at 37°C. Restriction products ran on 1% Agarose gel.

Gel results indicate ligation of pMaster+uMLP succeeded.

GEL IMAGE!!!

• Plasmids (pMaster+Survivin mCherry and pMaster+uMLP) were transformed into DH5α and inserted into 100ml of LB (+ampicillin) ON, for midiprep tomorrow.
• AAV Plasmids (Htert+SaCas9, Htert+dCas9-vp64, MLPE, SUBB) were transformed into DH5α and inserted into 100ml of LB (+ampicillin) ON, for midiprep tomorrow.
• Ligation of pMaster from 23.08 (vector) with uUBB (insert).

After ligation, products were transformed into DH5α and seeded on 2 LB agar plates with ampicillin ON.

27.08.15

In the lab today: Vlad, Shalev

• We performed midiprep. Nanodrop concentration checked.

	Nanodrop results
Htert+SaCas9	893ng/μl (1.86/2.29)
Htert+dCas9-vp64	688ng/μl (1.84/2.35)
MLPE	956ng/μl (1.86/2.28)
SUBB	349ng/μl (1.84/2.24)
pMaster+Survivin mCherry	450ng/μl (1.84/2.25)

• Colonies found on both plates of pMaster+uUBB from yesterday. 5X10ml of LB (+ampicillin) was inoculated with one colony each ON (3 colonies from plate 1, and 2 colonies from plate 2).

28.08.15

In the lab today: Vlad, Shalev

• We performed miniprep for pMaster+uUBB from yesterday

Then we perform Restrictions of with SalI, PstI (to check ligation). All restrictions done in incubation for 30 minutes at 37°C. Restriction products ran on 1% Agarose gel.

Gel results indicate that in the second colony of the first plate ligation of pMaster+uUBB succeeded.

GEL IMAGE!!!

• Restriction of DNA . All restrictions done in incubation for 30 minutes at 37°C. Then we run restriction products on 1% Agarose gel, extracted the appropriate DNA band (according to size) and performed gel extraction protocol.

	Restriction enzymes	Alkaline phosphatase
pMaster (1)	XhoI, BamHI	Added
pMaster (2)	SalI, XbaI	Added
dCas9-vp64	SalI, XbaI
SaCas9	XhoI, BamHI

GEL IMAGE!!!

29.08.15

In the lab today: Vlad, Shalev

• Ligations of restrictions from yesterday:

Ligation of pMaster (vector) with dCas9-vp64 (insert).

Ligation of pMaster (vector) with SaCas9 (insert).

After ligation, products were transformed into DH5α and seeded on LB agar plates with ampicillin ON.

• pMaster+uUBB (DH5α) glycerol stock was prepared.

30.08.15

In the lab today: Vlad, Shalev

• Colonies found on both plates (pMaster+dCas9-vp64 and pMaster+SaCas9) from yesterday. 10ml of LB (+ampicillin) was inoculated with one colony from each ON.
• We still had some restriction products of pMaster and dCas9-vp64 from 28.08, so we did Ligation of pMaster (vector) with dCas9-vp64 (insert) again. (in case ligation failed in current colonies). After ligation, products were transformed into DH5α and seeded on LB agar plates with ampicillin ON.

31.08.15

In the lab today: Vlad, Shalev

• Colonies found on pMaster+dCas9-vp64 plate from yesterday. 10ml of LB (+ampicillin) was inoculated with one colony ON.
• pMaster+Survivin mCherry (DH5α) glycerol stock was prepared.
• Restrictions of pMaster+dCas9-vp64 with SpeI, PacI (to check ligation). All restrictions done in incubation for 30 minutes at 37°C. Restriction products ran on 1% Agarose gel. Gel results indicate about a problem. Maybe PacI enzyme is not working as expected. We will try tomorrow other restriction enzymes set.
• Restrictions of pMaster+SaCas9 with AgeI, NotI (to check ligation). All restrictions done in incubation for 30 minutes at 37°C. Restriction products ran on 1% Agarose gel.

Gel results indicate ligation of pMaster+SaCas9 succeeded.

GEL IMAGE!!!

• pMaster +SaCas9 (DH5α) glycerol stock was prepared.

01.09.15

In the lab today: Vlad, Shalev

• Restrictions of pMaster+dCas9-vp64 from 29.08 and 30.08 (to check ligation). We decided to use different restriction enzymes because maybe PacI is problematic. So new enzymes are: SalI,XBaI. All restrictions done in incubation for 30 minutes at 37°C. Restriction products ran on 1% Agarose gel.

Gel results indicate ligation of pMaster+dCas9-vp64 from 29.08 succeeded.

GEL IMAGE!!!

• pMaster +dCas9-vp64 (DH5α) glycerol stock was prepared.
• 100ml of LB (+ampicillin) where made for midiprep. Each contained one of the following: pMaster+dCas9-vp64, pMaster+ SaCas9, pMaster+uMLP, pMaster+uUBB.

03.09.15

In the lab today: Vlad, Shalev

• We performed midiprep. Nanodrop concentration checked.

	Nanodrop results
pMaster +dCas9-vp64	1467ng/μl
pMaster +SaCas9	697ng/μl
pMaster +uMLP	1685ng/μl
pMaster +uUBB	1209ng/μl