Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/10/30

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Overview

Phusion gradient PCR.

Methods

Reaction volume: 160 µL (split into 8 tubes to be pooled later)

Reagent Stock Volume used (µL)
GC Buffer 5x 32
dNTP mix 10 mM 3.2
Primer (for) 100 µM 1.6
Primer (rev) 100 µM 1.6
Template 50 ng/µL 1.6
DMSO - 4.8
Phusion 20/µL 1.6
H2O - 113.6
Total 160

Reactions:

Reaction Forward primer Reverse primer template
EM7-ZeoR P121 P122 MV8
HPK-CFP P123 P124 DBN006

All primers have annealing temperature at 60°C

Thermal cycler program:

STEP TEMP TIME
Initial Denaturation 95°C 30 seconds
30 Cycles 95°C 10 seconds
50-70°C 20 seconds
72°C 60 seconds
Final Extension 72°C 10 minutes
Hold 4°C infinite

Results

Gel image:

2015-10-30 ZeoR CFP phusion PCR.jpg

Top lanes are ZeoR, bottom lanes are HPK-CFP. Annealing temperature does not appear to be a contributing factor. Must have been use of CG buffer or DMSO.

Next steps: PCR cleanup, maybe gel purification of HPK-CFP fragment (to avoid small fragment), LCR.