Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2016/01/08

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Overview

Running colony PCR on the HA2_v0120 ligation from yesterday and planning future steps.

Ligation Results

  • Negative control: ~250 tiny colonies
  • Treatment: ~1000 large colonies

Never seen this before... going to pick 8 colonies from the treatment and perform colony PCR using the v0120 primers.

Colony PCR

Prepare the following master mix in the first tube of an 8-tube PCR strip:

Reagent Volume (µL)
GoTaq 2x GRN80
P001 (10 µM)8
P002 (10 µM)8
H2O64

Mix all ingredients thoroughly then dispense 20 µL into each tube in the strip. Pick 6 colonies from the sample plate, and 2 colonies from the negative plate, using a fresh P20 tip for each one, and place in the reaction mix. Let sit for 10 minutes, then streak an LB-Amp plate split into 8 sections.

Colony PCR program:

Step Temp. °C Time (sec)
Initial denaturation95120
Denature9530
Anneal5530
Extension7230
Repeat x35
Final Extension72300
Soak4

Expected product size is 358 bp.

PCR Results

Looks like all 6 colonies picked from the ligation plate worked. Neither of the 2 negative control colonies show amplification in the expected size range. Next step: start liquid cultures from streak plate, plasmid miniprep, move on to next stage of assembly.


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