Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/08/05

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Overview

Trying to repeat the PCR for the first reaction using P197 & P215 primers (René's).

PCR

Reaction 1 (anneal temp. 54°C):

Reagent Volume (µL)
2x GoTaq MM 25
MB grade H2O 24
P197 (RD) 10 µM 0.25
P215 (RD) 10 µM 0.25
template 0.5
Step Temp. (°C) Duration (sec)
Initial Denature 95 120
Main cycle (x35):
denature 95 30
anneal 46-->54 30
extension 72 60
final extension 72 300
soak 4 indef.

Gel Electrophoresis Results

File:2015-08-05 Gal4EED-luc PCR products.TIF

Faint but visible bands at all temperature ranges (46-->54). Must have not put in one of the primers during the previous run. Will perform PCR clean-up and submit samples for sequencing in a couple days.