Tuesday 27^th September

Lab work

Visualization

PCR of extracted GFP 1.9 in pSB1C3 (pPS16_020)

By Maxence & Mahnaz

In order to verify if extracted plasmids contain GFP 1.9, a PCR was run with iPS168 & iPS169 to amplify the insert. For that purpose, plasmids sent for sequencing the 14th September (clones 2, 7, 8 and 12) and plasmids extracted the 23rd Sepembter (clones 3, 4, 7 and 8) were used.

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

• 1 µL of matrix
• 1 µL of dNTPs (10mM)
• 2.5 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	95°C	30sec
30 cycles	95°C	30sec
	64.4°C	30sec
	72°C	30sec
Final Extension	72°C	5min
Hold	4°C	$\infty$

Primers used were:

Matrix	Extracted plasmid pSB1C3 containing GFP 1.9 (pPS16_020)
Primers	iPS168 and iPS169

Gel of PCR products

By Maxence & Manhaz

4 µL of PCR products and 4 µL of DNA ladder were placed in wells and migrated at 100V during 30 min, in odrder to verify the DNA quantity.

PCR products expected were :

PCR products	Expected band size (bp)
GFP 1.9	1135

GEL

Colony PCR of 23 clones containing FKBP - GFP 10 in pSB1C3 (pPS16_018)

By Maxence, Mahnaz & Coline

As the two clones selected yesterday were not grown, a new colony PCR was done for 23 clones (16 clones obtained by Gibson with 2 fragments and 7 clones obtained by Gibson with 3 fragments) from the 23rd September. For that purpose, 23 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	30sec
Final Extension	72°C	7 min
Hold	4°C	$\infty$

Primers used were:

Matrix	Clones containing FKBP - GFP 10 in pSB1C3 (pPS16_19)
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)
FKBP - GFP 10 in pSB1C3 (pPS16_019)	1030

GEL