Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2016/01/28

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Overview

Using the QuikChange Site-Directed Mutagenesis Kit to fix the missense mutation in the ZeoR gene.

Prepare mixes (all units in µL):

Reagent Em7:ZeoR_v0120 (60 ng/µL) MV8 (500 ng/µL) control (pWhitescript)
10x Rxn Buffer 5 5 5
dsDNA template 1 0.2 2
125 ng for primer 2 2 1.25
125 ng rev primer 2 2 1.25
dNTP mix 1 1 1
H2O 38 38.8 38.5
PfuTurbo polymerase 1 1 1
Total 50 50 50

Cycle using the following parameters:

Segment Cycles Temperature Time
1 1 95 30 sec
2 12 95 30 sec
55 60 sec
68 4 min 30 sec
3 1 4 infinite

Tomorrow will treat with DpnI, transform into XL1-Blue competent cells.