User:Cassandra M Barrett/Notebook/Open Chromatin/2015/10/01
|Project name||Main project page|
Previous entry Next entry
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)
-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