Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/12/11

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Overview

Repeating PCR of pSB1A3_BsmBI fragment.

Reagent Stock Volume (µL)
GC Buffer5x10
dNTP mix10 mM1
DMSO-1.5
Phusion20 U/µL0.5
Template50 ng0.3
Primer (for)100 µM0.2
Primer (rev)100 µM0.2
H2O-36.3
Total50

Template: DBN001_pSB1A3 (close enough) Primers: P140, P141

PCR protocol: basic Phusion, 60°C annealing temp, 90 second extension time, 35 cycles.

Results

No amplification, because no binding site for reverse primer on selected template. Picked up some I13422_pSB1A3 from Wang Lab (pSB1A3 vector with pTet & GFP insert), that should work better. Will repeat attempt on Monday.


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