Two light blue colonies (there were no white colonies) were picked, restreaked on a new Kan50 plate (no X-gal) and PCR’d.

Master Mix:

2x Apex Red Master Mix – 37.5uL

10uM M13 Forward – 3uL

10uM M13 Reverse – 3uL

H2O – 46.5

Added 25uL to each PCR tube.

Cycling Params:

• 95C – 10mins

40 cycles of:

• 95C – 30s
• 55C – 30s
• 72C – 2mins

1 cycle:

• 72C – 10mins

Results:

The two pale blue colonies do NOT contain our desired insert. Despite the presence of a larger, faint band (~950bp), that is far smaller than our 5’RACE insert size (~1500bp). And, clearly, the primary amplicon produced is ~250bp, which is the expected size for empty vector… Ladder is Hyperladder I (Bioline).

Will need to re-do ligation reaction and will do so at the recommended volumes in the TOP TA (Invitrogen) protocol.