Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/11/25

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Overview

Ligation results, colony PCR & results, possibly submitting samples for sequencing.

Ligation Results

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.

Colony PCR

Prepare PCR tubes with the following mixture per tube:

Reagent Volume (µL)
GoTaq MM 2x 10
pJET forward (10 µM) 1
pJET reverse (10 µM) 1
H2O 8
Total 20

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.


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