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

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Sequence verification around tk-luc insert: PCR Main 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 MM25
MB grade H2O24
P197 (RD) 10 µM0.25
P215 (RD) 10 µM0.25
template0.5

Reaction 2 (anneal temp. 62°C):

Reagent Volume (µL)
2x GoTaq MM25
MB grade H2O24
P118 10 µM0.25
P119 10 µM0.25
template0.5


Step Temp. (°C) Duration (sec)
Initial Denature95120
Main cycle (x35):
denature9530
anneal54/6230
extension7260
final extension72300
soak4indef.

Gel Electrophoresis Results

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


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