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 Buffer5x32
dNTP mix10 mM3.2
Primer (for)100 µM1.6
Primer (rev)100 µM1.6
Template50 ng/µL1.6
DMSO-4.8
Phusion20/µL1.6
H2O-113.6
Total160

Reactions:

Reaction Forward primer Reverse primer template
EM7-ZeoRP121P122MV8
HPK-CFPP123P124DBN006

All primers have annealing temperature at 60°C

Thermal cycler program:

STEP TEMP TIME
Initial Denaturation95°C30 seconds
30 Cycles95°C10 seconds
50-70°C20 seconds
72°C60 seconds
Final Extension72°C10 minutes
Hold4°Cinfinite

Results

Gel image:

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.


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