Haynes Lab:Notebook/CRISPR Editing/2014/09/25

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Purified PCR product for Surveyor, P197,P198 forgot to save some unpurified on a gel

Sample Read# 260 280 260/280 ng/µL
Luc14 gDNA 0.131 0.071 1.844 130.862

Set up nested PCR with P199 and P200 of gRNA 20 samples and gRNA 29 samples and Luc14 genomic DNA (7 50ul reactions in triplicate to get enough)
Used 1ul of each template at 100-200ng/ul concentration for gRNA 20 samples. Used 1ul of 1:100 dilution of gRNA 29 samples and Luc14 gRNA control.

' 1rxn 21.5 rxns
H20 31.5 677.25
5x buffer 10 215
dNTPs 1 21.5
FP 2.5 53.75
RP 2.5 53.75
Add 145ul to each first tube for each set of reactions
add the following to each first tube
' 1rxn 3.053 rxns
template 1 3.053
DMSO 1.5 4.5795
Phusion 0.5 1.5265
split each first tube into the three tubes, 50ul each

Annealing Luc14 gDNA P197 P198

' ul for 400ng ul elution solution (3ul total) ul water ul 10x annealing buffer
gRNA 27 Luc 14 3.05 -0.05 6.00 1

Annealing protocol on wang lab machine
Digest with surveyor, follow same protocol from yesterday, 0.5ul nuclease for 45mins at 42deg
Added 1ul stop solution, kept on ice for about 5 minutes, ran on gel with the same amount of PCR product in the next lane.
Ran the nested PCR reactions on gel