Friday 16^th September

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Colony PCR of 16 clones containing FKBP - GFP 10 in pSB1C3 (pPS16_019)

By Mahnaz

Colonies were obtained for the Gibson done the 15th September. A colony PCR was done for 16 clones from the 15th September. For that purpose, 16 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 :

Result of the migration

Some PCR products were at the good size. Clones 7, 8, 9 and 10 were selected and were grown at 37°C overnight.