Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2016/02/08

Today's project is...

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Overview More adventures in cloning... To do: quantify DNA of the four DBN010 plasmid preps quantify DNA of HA1_v0120 plasmid (for future ligation) restriction digest of DBN010 (EcoRI & XbaI) gel purification of DBN010 (3700 bp fragment) quantify DNA of E&X digested DBN010 ligation: KAH184 (E&S) with DBN010 (E&X) transformation (quick) into DH5α-Turbo DNA Quantification quantification went fine, concentrations are between 50 and 70 ng/µL for DBN010. A bit lower for HA1_v0120, between 7 and 24 ng/µL. 260/280 values are good. Restriction Digest Reagent Volume (µL) DNA (DBN010) 25 FD 10x GRN 3 EcoRI 1 XbaI 1 Total 30 Incubate at 37 °C for 10 minutes, then purify via gel electrophoresis. Gel purification Gel image: All four plasmids appear to contain the insert. Will proceed with gel extraction from colonies 1 and 3. DNA Quantification, post-purification Concentration is ~7 ng/µL for both samples. 260/280 is on the high side, around 2.8-2.9. Going to proceed with ligation... Ligation Reagent Volume (µL) Negative Vector (50 ng) 7.1 7.1 Insert (2:1) 3.1 0.0 10x buffer 1.2 1.0 ligase 1.0 1.0 H2O 0.0 0.9 Total 12.4 10.0 Incubate at room temperature for 10 minutes, then take 1 µL from each sample (treatment & negative control) and use for quick transformation protocol.