User:Eric M. Walters/Notebook/Spring 2012/2012/06/04

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Continuing to enrich for the ΔacsA::barcode

A few things: despite all the evidence pointing to the 12 transformants I screened showing the proper insert, I'm skeptical since it was only 1:2::ctrl:transformant. I need to screen them off of the non-transformation plate with the BC-F and DnRv1 primers to be sure. My patches came up on 5 mM nicely, so I'll streak those onto more 5 mM AA for isolation, and hope to get some isolated colonies I can screen for purity.

Colony PCR of transformation streakouts

Colonies are from the initial 500 uM streakouts from the transformation, isolated. Two controls are gDNA (should yield nothing, lacking the barcode) and the linear DNA construct (should yield nothing, lacking the DnRv1 binding site).

Each 10 uL reaction:

  • 5 uL GoTaq
  • .5 uL BC-F
  • .5 uL acsA-DnRv1
  • 4 uL MilliQ water

52 degree annealing temp, :40 extension.

13 colonies total, the 13th being #14, which was on the negative control plate.

Pflegerlab 2012-06-04 15hr 47min colony PCR of 2nd gen acsA;;BC.jpg