Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2016/01/18

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Overview

Going over ligation results, performing colony PCR and growing up liquid cultures for plasmid prep.

Ligation & Transformation Results

Negative control: ~200 small colonies
Ligation plate: ~200 small colonies, plus ~10 large colonies

Going to pick 8 of the large colonies for colony PCR & liquid culture.

Colony PCR

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

Reagent Volume (µL)
GoTaq 2x GRN 80
P001 (10 µM) 8
P002 (10 µM) 8
H2O 64

Mix all ingredients thoroughly then dispense 20 µL into each tube in the strip. Pick 8 colonies from the sample plate using a fresh P20 tip for each one, and place in the reaction mix. Let sit for 10 minutes, then transfer tips to 5 mL of liquid culture and start shaking at 37 °C.

Colony PCR program:

Step Temp. °C Time (sec)
Initial denaturation 95 120
Denature 95 30
Anneal 55 30
Extension 72 30
Repeat x30
Final Extension 72 300
Soak 4

Expected product size is 701 bp.

Results:

File:2016-01-18 EM7-ZeoR v0120 ligation results v2.tif

Looks like cultures 1, 2, 4, 5, 7, and 8 contain the insert. Will proceed with these for plasmid prep & sequencing.

Plasmid Prep

Used the Sigma Plasmid Miniprep kit.

DNA Quantification

All six samples are between 50-60 ng/µL in concentration, with good 260/280 values.

Submitting three of the samples for sequencing tomorrow.