Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/11/04

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Overview

Phusion PCR on LCR fragment.

Reaction volume: 100 µL (split into 4 tubes to be pooled later)

Reagent Stock Volume used (µL)
GC Buffer 5x 20
dNTP mix 10 mM 2
Primer (for) 100 µM 1
Primer (rev) 100 µM 1
Template 50 ng/µL 15
DMSO - 3
Phusion 20/µL 1
H2O - 57
Total 100

Reactions:

Reaction Forward primer Reverse primer template
DBN008 P40 P41 LCR product

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
60°C 20 seconds
72°C 60 seconds
Final Extension 72°C 10 minutes
Hold 4°C infinite

Results

2015-11-04 PCR of LCR annotated.png

Nonspecific binding and amplification in the sample. Not sure what's going wrong here... this is after PCR of the LCR product, using forward primer for HA1 and reverse primer for HA2. Product size should be approximately 2600-2700bp