Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/03/24

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Overview

I cut out the 1500bp band in lane 8 from the agarose gel run yesterday and purified the DNA using the Sigma GenElute Gel Extraction Kit (protocol can be viewed here). After extracting the DNA, I performed a thermal gradient PCR on the fragment using the following quantities of reagents:

Reagent Volume (uL) for 9 rxns (uL)
2x GoTaq MM 10 90
MB grade H2O 9.6 86.4
forward primer 0.1 0.9
reverse primer 0.1 0.9
template 0.2 1.8
Total 20 180

Thermal cycler program:

Step Temperature °C Duration
Initial denature 95 120 sec
denature 95 30 sec
anneal 59-->66 30 sec
extension 72 90 sec
final extension 72 300 sec
soak 4 indef.

Thermal gradient is from 59°C to 66°C. Program name is DBN20150324.