Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/07/22

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Owwnotebook icon.pngSequence verification around tk-luc insert: PCR Report.pngMain project page
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Overview

In order to verify that the luc replacement is successful we will have to sequence the region around the gene before and after using primers specific to the old and new inserts. I previously extracted genomic DNA from Gal4EED-luc HEK293 cell line and will be performing PCR to amplify the regions immediately upstream and downstream of the insert.

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

Reaction 2 (anneal temp. 62°C):

Reagent Volume (µL)
2x GoTaq MM 25
MB grade H2O 24
P118 10 µM 0.25
P119 10 µM 0.25
template 0.5


Step Temp. (°C) Duration (sec)
Initial Denature 95 120
Main cycle (x35):
denature 95 30
anneal 54/62 30
extension 72 60
final extension 72 300
soak 4 indef.

Gel Electrophoresis Results

File:2015-07-22 Gal4EED-luc PCR products.TIF

2nd reaction worked, 1st did not. Will try and repeat using lower temperature and gradient.