Thuersday 22^nd September

Lab work

Visualization

Colony PCR of 8 clones containing FRB - GFP 11 - GFP 1.9 in pSB1C3 (pPS16_022)

By Maxence, Mahnaz & Coline

A colony PCR was done for 8 clones (4 clones with GFP 1.9 PCR product obtained with DMSO and 4 clones with GFP 1.9 PCR product obtained without DMSO) from the 20th September. For that purpose, 8 clones were screened and used for the usual protocol of Colony PCR. The same colonies were also plated on Petri dish containing solid LB + 30 µg/mL Cm and liquid cultures (LB + 30 µg/mL Cm) were made from these clones. Both petri dishes and liquid cultures were grown at 37°C.

For each clones contained in 20 μl water, 5.13 μL of the following mix were added :

• 2.5 µL DreamTaq Buffer
• 0.5 µL of dNTPs (10mM)
• 1 µL of each primer mix (10µM)
• 0.13 μl of DreamTaq Pol

PCR was performed as follow:

Step	Temperature	Time
Initial denaturation	95°C	3 min
30 cycles	95°C	30 sec
	48.4°C	30 sec
	72°C	1min 30sec
Final Extension	72°C	7 min
Hold	4°C	$\infty$

Primers used were:

Matrix	Clones containing FRB - GFP 11 - GFP 1.9 in pSB1C3 (pPS16_022)
Primers	iPS168 and iPS169

After amplification, 3 µL of each PCR products and 5 µL of DNA ladder were placed in wells and migrated at 100V during 30 min.

PCR products expected were :

PCR products	Expected band size (bp)
FRB - GFP 11 - GFP 1.9	1800

GEL

PCR products were not at the good size.

Colony PCR of 12 clones containing FKBP - GFP 10 in pSB1C3 (pPS16_019)

By Maxence, Mahnaz & Coline

As sequencing results were not convincing, a new colony PCR was done for 12 clones from the 15th September (8 new clones and the 4 sent for sequencing) but with primers iPS168 & iPS169. For that purpose, 12 were screened and used for the usual protocol of Colony PCR. The same colonies were also plated on Petri dish containing solid LB + 30 µg/mL Cm and liquid cultures (LB + 30 µg/mL Cm) were made from these clones. Both petri dishes and liquid cultures were grown at 37°C.

For each clones contained in 20 μl water, 5.13 μL of the following mix were added :

• 2.5 µL DreamTaq Buffer
• 0.5 µL of dNTPs (10mM)
• 1 µL of each primer mix (10µM)
• 0.13 μl of DreamTaq Pol

PCR was performed as follow:

Step	Temperature	Time
Initial denaturation	95°C	3 min
30 cycles	95°C	30 sec
	48.4°C	30 sec
	72°C	30 sec
Final Extension	72°C	7 min
Hold	4°C	$\infty$

Primers used were:

Matrix	Clones containing FKBP - GFP 10 in pSB1C3 (pPS16_19)
Primers	iPS83 and iPS84

After amplification, 3 µL of each PCR products and 5 µL of DNA ladder were placed in wells and migrated at 100V during 30 min.

PCR products expected were :

PCR products	Expected band size (bp)
FKBP - GFP 10 in pSB1C3 (pPS16_019)	757

GEL

PCR products were not at the good size, plasmids were empty.

PCR on cleaned up PCR products FKBP from 8th September and gblock 2.2 from the 8th September in order to fuse fuse these fragments

By Maxence, Manhaz & Coline

As sequencing and colony PCR of clones containing FKBP - GFP 10 in pSB1C3 did not show good results, a new clonage strategy was performed: before the Gibson, the two inserts were fused together by PCR. So PCR was performed with cleaned up PCR products FKBP (obtained the 8th September) and claned up PCR product gblock 2.2 (obtained the 8th September) with the following protocol.

For each 50μl of reaction, mix the following reagents :

• 1 µL of each PCR product
• 1 µL of dNTPs (10mM)
• 2 µL of each primer mix (10µM)
• 10 µL of buffer (5X)
• 0,5 µL of Phusion polymerase
• 32.5 µL of nuclease free water

Mix gently and aliquot 50 μl of the mix into the PCR tubes on ice. Perform PCR as follow:

Step	Temperature	Time
Initial denaturation	98°C	30sec
30 cycles	98°C	10sec
	65°C	30sec
	72°C	20sec
Final Extension	72°C	7min
Hold	4°C	$\infty$

Primers used were:

Matrix	PCR products FKBP from 8th September and gblock 2.2 from the 8th September
Primers	iPS145 and iPS84

Gel of PCR products

By Maxence, Mahnaz & Coline

After amplification, 3 µL of each cleaned up PCR products and 5 µL of DNA ladder were placed in wells and migrated at 100V during 30 min.

PCR products expected were :

PCR products	Expected band size (bp)
FKBP - GFP 10	747

GEL

PCR product was at the good size.

Colony PCR of 8 clones containing corrected dCas9 ST - GFP 11 in pSB1C3 (pPS16_017)

By Maxence, Mahnaz & Coline

A colony PCR was done for 8 clones from the 20th September. For that purpose, 8 clones were screened and used for the usual protocol of Colony PCR. The same colonies were also plated on Petri dish containing solid LB + 30 µg/mL Cm and liquid cultures (LB + 30 µg/mL Cm) were made from these clones. Both petri dishes and liquid cultures were grown at 37°C.

For each clones contained in 20 μl water, 5.13 μL of the following mix were added :

• 2.5 µL DreamTaq Buffer
• 0.5 µL of dNTPs (10mM)
• 1 µL of each primer mix (10µM)
• 0.13 μl of DreamTaq Pol

PCR was performed as follow:

Step	Temperature	Time
Initial denaturation	95°C	3 min
25 cycles	95°C	30 sec
	48.4°C	30 sec
	72°C	4min
Final Extension	72°C	7 min
Hold	4°C	$\infty$

Primers used were:

Matrix	Clones containing FRB - GFP 11 - GFP 1.9 in pSB1C3 (pPS16_022)
Primers	iPS168 and iPS169

Colony PCR of 8 clones containing GFP 1.9 in pSB1C3 (pPS16_020)

By Maxence, Mahnaz & Coline

As sequencing results were not good, a new colony PCR was done for 8 clones from the 12th September. For that purpose, 8 clones were screened and used for the usual protocol of Colony PCR. The same colonies were also plated on Petri dish containing solid LB + 30 µg/mL Cm and liquid cultures (LB + 30 µg/mL Cm) were made from these clones. Both petri dishes and liquid cultures were grown at 37°C.

For each clones contained in 20 μl water, 5.13 μL of the following mix were added :

• 2.5 µL DreamTaq Buffer
• 0.5 µL of dNTPs (10mM)
• 1 µL of each primer mix (10µM)
• 0.13 μl of DreamTaq Pol

PCR was performed as follow:

Step	Temperature	Time
Initial denaturation	95°C	3 min
30 cycles	95°C	30 sec
	48.4	30 sec
	72°C	30sec
Final Extension	72°C	7 min
Hold	4°C	$\infty$

Primers used were:

Matrix	Clones containing GFP 1.9 in pSB1C3 (pPS16_020)
Primers	iPS168 and iPS169

Gel of PCR products

By Maxence, Mahnaz & Coline

After amplification, 3 µL of each PCR products and 5 µL of DNA ladder were placed in wells and migrated at 100V during 30 min.

PCR products expected were :

PCR products	Expected band size (bp)
dCas9 ST - GFP 11	3800
GFP 1.9 in pSB1C3	862

GEL

For GFP 1.9 in pSB1C3, all PCR products were at the good size, clones X, X, X and X were selected for sequencing. For dCas ST - GFP 10, 6 PCR products were at the good size, clones X, X, X and X were selected for sequencing.