Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/11/25
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Ligation results, colony PCR & results, possibly submitting samples for sequencing.
Treatment (LCR product & pJET): ~25 colonies Negative control (pJET only): 4 colonies
Looks like the ligation worked. Will pick 8 colonies and proceed with colony PCR.
Prepare PCR tubes with the following mixture per tube:
Label tubes 1-8. For each tube, pick 1 colony from the ligation plate using a 20 µL pipette tip and place the tip directly into the PCR tube. Allow the tubes & tips to incubate for 10 minutes, then streak a labeled LB-Amp plate with the tips. Set up PCR using the lab thermal cycler with annealing temperature of 58°C.
Colony PCR Results
No insert detected in any of the 8 colonies picked. Going to assume this was a failure. Not proceeding to sequencing.