Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/10/28

From OpenWetWare

Jump to: navigation, search
Phusion PCR of parts for LCR Main project page
Previous entry      Next entry

Overview

We're using the Phusion polymerase to create excess template in anticipation of performing ligase-cycling reaction (LCR) in the near future. This will allow us to incorporate a zeocin resistance (ZeoR) gene upstream of the HPK-CFP fluorescent reporter.

Methods

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

Reagent Stock Volume used (µL)
HF Buffer5x20
dNTP mix10 mM2
Primer (for)100 µM1
Primer (rev)100 µM1
Template50 ng/µL1
Phusion20/µL1
H2O-74
Total100

Reactions:

Reaction Forward primer Reverse primer template
HA1 (1)P40P120DBN007
EM7-ZeoR (2)P121P122MV8
HPK-CFP (3)P123P124DBN007
HA2 (4)P125P41DBN007

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



Personal tools