Colony PCR - 5’ RACE Colony: COX2

One white colony (marked with arrow in image linked) from the two plates from Steven’s cloning (from yesterday) was picked, restreaked on a new Kan50 plate (no X-gal) and PCR’d.

Master Mix:

2x Apex Red Master Mix - 25uL

10uM M13 Forward - 1uL

10uM M13 Reverse - 1uL

H2O - 23

Added 25uL to each PCR tube.

Cycling Params:

  • 95C - 10mins

40 cycles of:

  • 95C - 30s

  • 55C - 30s

  • 72C - 2mins

1 cycle:

  • 72C - 10mins


Lane 1: Hyperladder IV

Lane 2: colony PCR

Lane 3: NTC

Turns out the Hyperladder IV (this gel was run in the Friedman Lab) only goes up to 1000bp. So, the band we see in the colony PCR reaction could be close to the expected size if the insert is present (~1500bp). Although, we also see a band in the NTC. Will repeat this PCR and run on a gel with a more appropriate ladder…