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: 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.