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

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PCR of DBN006, adding IIS sites Main 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 mix25
MB grade H2O24
DBN003 f1 primer0.25
DBN002 r1 primer0.25
template0.5
Total50
Step Temperature °C Duration
Initial denature95120 sec
Main Cycle (x35):
denature9530 sec
anneal4830 sec
extension72120 sec
final extension72300 sec
soak4indef.

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.


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