Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2016/01/18
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Going over ligation results, performing colony PCR and growing up liquid cultures for plasmid prep.
Ligation & Transformation Results
Negative control: ~200 small colonies
Going to pick 8 of the large colonies for colony PCR & liquid culture.
Prepare the following master mix in the first tube of an 8-tube PCR strip:
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:
Expected product size is 701 bp.
Looks like cultures 1, 2, 4, 5, 7, and 8 contain the insert. Will proceed with these for plasmid prep & sequencing.
Used the Sigma Plasmid Miniprep kit.
All six samples are between 50-60 ng/µL in concentration, with good 260/280 values.
Submitting three of the samples for sequencing tomorrow.