Haynes Lab:Notebook/CRISPR Editing/2014/09/17

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09/17/2014

Added 1ul Enhancer and 1ul Surveyor nuclease on ice to the annealing reactions from yesterday. Incubate for 60 min at 42 in thermal cycler. Ran on 1% gel:



Trying to increase sample genomic DNA PCR product concentration. Varied starting template concentration and reaction volume:

Rxn # template uL water 5x phusion HF buffer 10mM DNTPs 10uM f primer 10uM r primer Template DMSO Phusion Total rxn V
1 gRNA 20 +dox rep 1 10.8 4 0.4 1 1 2.00 0.6 0.2 20.0
2 gRNA 20 +dox rep 1 30.0 10 1 2.5 2.5 2.00 1.5 0.5 50.0
3 gRNA 20 +dox rep 1 8.8 4 0.4 1 1 4.00 0.6 0.2 20.0
4 gRNA 20 +dox rep 1 28.0 10 1 2.5 2.5 4.00 1.5 0.5 50.0