Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/05/06

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Owwnotebook icon.pngPCR of DBN006, adding IIS sites Report.pngMain project page
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Overview

Today I'll be performing PCR on the DBN006 plasmids I validated yesterday (from colonies 1 and 3), adding IIS restriction enzyme cut sites on their ends so they can be inserted into the DBN001 fragment containing homology arms.

PCR Overview

Reagent Volume (uL)
2x GoTaq master mix 25
MB grade H2O 24
DBN003 f1 primer 0.25
DBN002 r1 primer 0.25
template 0.5
Total 50
Step Temperature °C Duration
Initial denature 95 120 sec
Main Cycle (x35):
denature 95 30 sec
anneal 48 30 sec
extension 72 120 sec
final extension 72 300 sec
soak 4 indef.

Once PCR is complete, take 27 µL of each product, add 3 µL of 10x loading dye and verify product size via gel electrophoresis. If band matches, proceed with gel purification.