User:Anthony Salvagno/Notebook/Research/2009/03/25/Toyoko Miniprep Protocol
Toyoko Miniprep Protocol
Instead of using columns we did a quick protocol provided by Toyoko:
- Add 0.5ml overnight culture to 0.5ml phenol/chloroform; keep the remaining culture at 4C until we know result
- Vortex 1 minute
- Spin 12krpm for 5 min.
- Take 0.45ml top layer and mix with 0.9ml 100% cold Ethanol
- Mix and spin immediately (no need to wait 60' in this case) at 12krpm for 5 minutes
- Pour off supernatant; wash pellet with 500ul 70% Ethanol
- Dry pellet and resuspend in 20ul 1xTE plus 20ug/ml RNaseA; incubate 37C for 60 minutes
- Mix 5ul DNA prep with 3ul 5xLoading dye and 10ul H2O, save remaining DNA samples for RE digests if we ID inserts
- Load onto 1% agarose gel in TAE; Also load 250ng uncut pRS413 vector to compare
- Vectors with insert should migrate more slowly than vector control.