Haynes Lab:Notebook/CRISPR Editing/2016/03/12: Difference between revisions
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==03/12/2016== | ==03/12/2016== | ||
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. <br>Ran on gel | 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. <br>Ran on gel<br> | ||
[[image:2016-03-12_colony_PCR.png|420px]]<br> | |||
Colonies 1, 3, 4, 5, and 6 look correct. I should mini prep those five for sequencing | |||
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Miniprepped g031 in pX330g | Miniprepped g031 in pX330g | ||
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Ran gDNA extractions on gel since I left them lysing at 56deg for 18 hours | Ran gDNA extractions on gel since I left them lysing at 56deg for 18 hours<br> | ||
[[image:GDNA_gel_2016-03-12_19.57.31.png|420px]]<br> | |||
Looks like they're still good, can PCR them tomorrow woo | |||
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Moved cells to T25 and T75 flask | Moved cells to T25 and T75 flask | ||
Revision as of 20:05, 12 March 2016
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03/12/2016Colony 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.
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