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

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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 Buffer 5x 20
dNTP mix 10 mM 2
Primer (for) 100 µM 1
Primer (rev) 100 µM 1
Template 50 ng/µL 1
Phusion 20/µL 1
H2O - 74
Total 100

Reactions:

Reaction Forward primer Reverse primer template
HA1 (1) P40 P120 DBN007
EM7-ZeoR (2) P121 P122 MV8
HPK-CFP (3) P123 P124 DBN007
HA2 (4) P125 P41 DBN007

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