Thuersday 25^th September

Lab work

Visualization

Colony PCR of 12 colonies containing plasmid pJET coding sg-Nm and 12 colonies containing plasmid pJET coding FRB

Mahnaz

Transformed cells which had been grown overnight were used for colony PCR. For that purpose, 12 clones containing sg-Nm and 12 clones containing FRB were screened and used for the usual protocol of Colony PCR. The same colonies were also plated on Petri dish containing solid LB + 50 µg/mL Amp and liquid cultures (LB + 50 µg/mL Amp) were made from these clones. Both petri dishes and liquid cultures were grown at 37°C.

For each clones contained in 20 μl water, 4.13 μL of the following mix were added :

• 2.5 µL DreamTaq Buffer
• 0.5 µL of dNTPs (10mM)
• 0.5 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

GEL GEL GEL

We can't see anything, the only visibles strands are the priers sequence, The result is not satisfying