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 Buffer555
dsDNA template10.22
125 ng for primer221.25
125 ng rev primer221.25
dNTP mix111
H2O3838.838.5
PfuTurbo polymerase111
Total505050

Cycle using the following parameters:

Segment Cycles Temperature Time
119530 sec
2129530 sec
5560 sec
684 min 30 sec
314infinite

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


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