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

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DBN002 Gel Extraction & PCR Main project page
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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 MM1090
MB grade H2O9.686.4
forward primer0.10.9
reverse primer0.10.9
template0.21.8
Total20180

Thermal cycler program:

Step Temperature °C Duration
Initial denature95120 sec
denature9530 sec
anneal59-->6630 sec
extension7290 sec
final extension72300 sec
soak4indef.

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



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