User:Cassandra M Barrett/Notebook/Open Chromatin/2015/09/17

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Phusion PCR for LCR Parts

Purpose: to amplify ATF2 and Gal4DB-mCh parts for insertion into MV10. I am repeating this PCR as a check, old parts were discarded.

Methods:

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

1 reaction:

  • 05.uL 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

All primers were diluted to 10uM for a 100uM stock by adding 5uL of stock to 45uL of PCR water

PCR was run on standard Phusion protocol with an annealing temperature of 56C (primer TMs were rather different so this could be a good reason for the PCR not to work...) and an elongation time of 30 seconds

Results:

2uL of each PCR product was run on a 1% agrose gel for 35 minutes at 110V

Image:LCR_Parts.jpg

Conclusions:

Both parts appear to be appropriate lengths based on what is expected. Parts will be purified with Qiagen PCR clean up kit.



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