Wednesday 5^th October

Visualization

Colony PCR of 13 clones containing GFP 1.9 in pSB1C3 (pPS16_020)

By Maxence & Yacine

For that purpose, 13 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:

Primers used were:

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 :

No PCR products were obtained.

Plasmids extraction of FRB - GFP 11 in pSB1C3 (pPS16_019) clone 4 and FKBP - GFP 10 in pSB1C3 (pPS16_018) clone 6

By Maxence & Yacine

The following plasmids were extracted using a "standard Plasmid Miniprep":

• pPS16_018 (FKBP - GFP 10) clone 6
• pPS16_019 (FRB - GFP 11) clone 4

Cloning of FRB - GFP 11 (pPS16_019) in FKBP - GFP 10 - pSB1C3 (pPS16_018) by digestion-ligation

By Maxence & Yacine

For that purpose, FRB - GFP 11 (pPS16_019) clone 4 was cut by restriction enzymes XbaI & PstI as following:

• 20 µL of FRB - GFP 11 (pPS16_019) clone 4
• 4 µL of buffer FD
• 2 µL of restriction enzyme XbaI
• 2 µL of restriction enzyme PstI
• 10 µL of water

And FKBP - GFP 10 - pSB1C3 (pPS16_018) clone 6 was cut by restriction enzymes PstI & XbaI and was treated by the alkaline phosphatase FastAP (dephosphorylation of cloning vector DNA to prevent recircularization during ligation) as following:

• 20 µL of FKBP - GFP 10 - pSB1C3 (pPS16_018) clone 6
• 4 µL of buffer FD
• 2 µL of restriction enzyme SpeI
• 2 µL of restriction enzyme PstI
• 2 µL of alkaline phosphatase FastAP
• 10 µL of water

The mix were incubated for 20 minutes at 37°C. Then, 40 µL of each digestion products and 5 µL of DNA ladder were placed in wells and migrated at 100V during 30 min.

Migration products expected were :

Result of the migration

The digestion products were cleaned up from the gel by using the NucleoSpin Gel and PCR Clean-up kit protocol.