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

Sequence verification around tk-luc insert: PCR

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