Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/11/12
|Today's project is...||Main project page|
Previous entry Next entry
Transformation and colony PCR.
Follow the ligation protocol outlined in Assembly 101.
chemically competent cells & ligation protocol.
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.
Colony 2 looks like it might have the right product... rest look negative. Will submit for sequencing once I can do plasmid miniprep.