Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/12/15

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Overview

Phusion PCR of HPK:CFP part.

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) 10 µM 2
Primer (rev) 10 µM 2
H2O - 32.7
Total 50

Template: DBN007
Primers: P147, P151

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

Gel Results

File:2015-12-16 ZeoR and HA1 restriction digest and HPK-CFP PCR.tif

My PCR product is on the far right. Lots of non-specific binding and amplifcation. Main bands are at ~1500bp and ~2000bp. Expected product size is 2000bp.