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

From OpenWetWare

Jump to: navigation, search
Today's project is... Main project page
Previous entry      Next entry

Overview

Phusion PCR on LCR fragment.

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

Reagent Stock Volume used (µL)
GC Buffer5x20
dNTP mix10 mM2
Primer (for)100 µM1
Primer (rev)100 µM1
Template50 ng/µL15
DMSO-3
Phusion20/µL1
H2O-57
Total100

Reactions:

Reaction Forward primer Reverse primer template
DBN008P40P41LCR product

All primers have annealing temperature at 60°C

Thermal cycler program:

STEP TEMP TIME
Initial Denaturation95°C30 seconds
30 Cycles95°C10 seconds
60°C20 seconds
72°C60 seconds
Final Extension72°C10 minutes
Hold4°Cinfinite

Results

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


Personal tools