PCR – Colony Screening

Eight white colonies were selected for PCR screening to verify that they indeed contain the AF133090 insert, using M13 vector primers. Master mix calcs are here. Using sterile toothpicks, colonies were picked, re-streaked and then used to “inoculate” the PCR reactions. Cycling params were as follows:

1 cycle of:

  • 95C – 10m

40 cycles of:

  • 95C – 15s
  • 55C – 15s
  • 72C – 2m

PCR reactions were run on 0.8% TBE agarose gel.

Results:

Gel Loading:

Lane 1 – Hyperladder II (Bioline)

Lane 2 – Colony 1

Lane 3 – Colony 2

Lane 4 – Colony 3

Lane 5 – Colony 4

Lane 6 – Colony 5

Lane 7 – Colony 6

Lane 8 – Colony 7

Lane 9 – Colony 8

Lane 10 – NTC

The prominent bands seen on the gel all run at the expected size (~1600bp). Will select one of these re-streaked colonies for mini prep on Monday.

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