User:Cassandra M Barrett/Notebook/Open Chromatin/2015/10/02

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Preparation of inserts for LCR

Purpose: to amplify ATF2 and Gal4DB-mCh parts for insertion into MV10. I am repeating this PCR to get higher concentrations of inserts. Phusion didn't amplify well yesterday.

Methods:

Set up 4 reactions (ATF2, Gal4DB_mCh, and negative controls)

1 reaction:

  • 0.5uL template
  • 1uL 10uM FP
  • 1uL 10uM RP
  • 25uL 2X GoTaq Mastermix
  • 22.5uL Water

-Template for ATF2: ATF2 mp -FP for ATF2: ATF2 F1 -RP for ATF2: ATF2 R2

-Template for Gal4DB_mCh: KAH228 mp -FP for Gal4DB_mCh: Gal4DB F2 -RP for Gal4DB_mCh: mCh R1

PCRs were run on standard GoTaq protocol (annealing temp: 52C, elongation time: 1min)


Results:

Gel shows proper band sizes, both extracted and purified. Good yields!



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