Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/11/12

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Overview

Transformation and colony PCR.

Methods

Ligation

Follow the ligation protocol outlined in Assembly 101.


Transformation

chemically competent cells & ligation protocol.


Colony PCR

Dozens of colonies on both the ligation plate and negative control (vector only). Picked 8 colonies from the ligation plate. For colony PCR: Made a GoTaq master mix using 2X GoTaq Green, forward & reverse primers, and aliquoted 20 µL into 8 tubes. Picked 8 colonies and placed the micropipette tips in the tubes for 10 mins, then streaked plates. Performed PCR on the tubes containing bacteria + master mix.

Results

Gel:

File:2015-11-13 DBN008 pSB1A3 colony PCR check.tif

Colony 2 looks like it might have the right product... rest look negative. Will submit for sequencing once I can do plasmid miniprep.