User:Cassandra M Barrett/Notebook/Open Chromatin/2015/10/01: Difference between revisions
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==Preparation of inserts for | ==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. | Purpose: to amplify ATF2 and Gal4DB-mCh parts for insertion into MV10. I am repeating this PCR to get higher concentrations of inserts. | ||
Revision as of 09:18, 2 October 2015
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Preparation of inserts for LCRPurpose: 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:
-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
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