Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/12/14

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Overview

Repeating PCR of pSB1A3, this time with the correct template.

Materials/Methods

Reagent Stock Volume (µL)
GC Buffer 5x 10
dNTP mix 10 mM 1
DMSO - 1.5
Phusion 20 U/µL 0.5
Template 50 ng 0.3
Primer (for) 100 µM 0.2
Primer (rev) 100 µM 0.2
H2O - 36.3
Total 50

Template:I13522_pSB1A3 Primers: P140, P141

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

Results

File:2015-12-14 restriction digest and PCR product gels.tif

See rightmost lane. Band visible in the correct size range. Looks like the PCR worked.

Performed PCR cleanup on pSB1A3_BsmBI, measured concentration using Take3 plate reader. Final concentration: 31.4 ng/µL