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.


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


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