Haynes Lab:Notebook/CRISPR Editing/2016/03/12

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Colony PCR of colonies on ligation plates from Saturday. Primers P220/221 will give a 1240bp band if GFP is inserted. will give a 439bp for backbone only. Will be slightly longer if the backbone was only cut once. Used 2x taq MM. NEB suggests an annealing T of 51°C with Taq, will do 55°C to try to avoid lots of off target binding to gDNA.
Ran on gel
2016-03-12 colony PCR.png
Colonies 1, 3, 4, 5, and 6 look correct. I should mini prep those five for sequencing

Miniprepped g031 in pX330g

Ran gDNA extractions on gel since I left them lysing at 56deg for 18 hours
GDNA gel 2016-03-12 19.57.31.png
Looks like they're still good, can PCR them tomorrow woo

Moved cells to T25 and T75 flask