User:Cassandra M Barrett/Notebook/Open Chromatin/2015/10/01

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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.

Methods:

Set up 3 reactions (ATF2, Gal4DB_mCh, and negative control using Gal4DB_mCh primers)

1 reaction:

  • 0.5uL template
  • 1uL 10uM FP
  • 1uL 10uM RP
  • 1uL 10uM dNTPs
  • 0.5uL Phusion Pol
  • 10uL 5X HF Buffer
  • 36uL 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

PCR was run on standard Phusion protocol with an annealing temperature of 56C and an elongation time of 30 seconds

Results:

ATF2 did not amplify properly (only plasmid on gel?), Gal4DB_mCh amplified properly, purify with Qiagen PCR clean up kit. Reattempt ATF2 amplification tomorrow.

Gal4DB_mCh: 11ng/uL...probably re do this one also with more appropriate separate annealing temps