Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2016/02/09

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Overview DBN011 ligation results: Negative control: 8 colonies Treatment: 18 colonies Going to pick 4 colonies from the treatment plate and proceed with streaks & liquid cultures. To do: plasmid prep DNA quantification Restriction digest, HA1_v0120 with E&S (106 bp) Restriction digest, DBN011 with E&X (5769 bp) DNA quantification ligation transformation Plasmid preps HA1_v0120 1: 24 ng/µL HA1_v0120 2: 7 ng/µL DBN011 1: 124 ng/µL DBN011 2: 106 ng/µL DBN011 3: 27 ng/µL DBN011 4: 96 ng/µL Restriction Digests HA1 (E&S): Reagent Volume (µL) DNA 25 FD 10x GRN 3 EcoRI 1 Spe1 1 Total 30 DBN011 (E&X): Reagent Volume (µL) DNA 25 FD 10x GRN 3 EcoRI 1 XbaI 1 Total 30 Incubate at 37 °C for 10 minutes, then gel purify. HA1 part is 106 bp, DBN011 part is 5769 bp. Gel Extraction Results HA1 part looks good, but very faint band. DBN011 1, 2, and 4 look good. Proceeding to extract 1 and 2. HA1 (E&S digested): 4 ng/µL, 260/280 high (~3) DBN011 1 (E&X digested): 18.1 ng/µL DBN011 2 (E&X digested): 13.6 ng/µL Ligation Reagent Volume (µL) Negative Vector (DBN011 1) (50 ng) 2.8 2.8 Insert (HA1) (10:1) 2.3 0.0 10x buffer 1.0 1.0 ligase 1.0 1.0 H2O 2.9 5.2 Total 10.0 10.0 Incubate at room temperature for 10 minutes, then take 1 µL of reaction volume and use for quick transformation. Count colonies tomorrow.