Haynes Lab:Notebook/CRISPR Editing/2015/07/09

Project name

Main project page Previous entry      Next entry

07/09/2015 Purified nested PCR products from 7/8 Luc14 1.0ug 0.1ug 0.01ug, Gal4-EED+puro 1.0ug Gal4-EED+dox 1.0ug 0.1ug 0.01ug Ran surveyor on 400ng template in 15ul total. Annealed using manufacturer's protocol, 1ul enhancer, 1ul of surveyor enzyme, incubate at 42°C for 1 hour. add stop solution, dilute 1:20 in water, run on bioanalyzer. Results Luc14 samples were first and I saw some random cut bands that weren't in the no-enzyme control (so definitely not background amplification). Tried adding Mg to the next round of samples but saw the same bands. Unclear what those could mean. Consider redoing PCR. siRNA samples are not dense enough to do anything with. Will add media with puro to prevent re-silencing and let them grow over the weekend.