Plasmid Miniprep
- Pick one colony into 5 ml LB + antibiotic selection (50 µg/ml ampicillin -- If plasmid is larger than ~12 Kb, double the amount of antibiotic) and grow O/N at 37°C.
- Spin down 1.5 ml of overnight culture in Eppendorf for 1 minute on High.
- Asprirate supernatant and resuspend cell pellet in 100 µl Solution I.
- Add 200 µl Solution II mix gently by inversion 10-15 times.
- Add 150 µl Solution III, vortex briefly to mix and spin for 5 minutes on High.
- Transfer supernatant to fresh tube and add 500 µl phenol:chloroform, vortex and spin for 5 minutes on High.
- Transfer aqueous layer to fresh tube and add 1 ml ethanol, mix well by inversion and spin for 5 minutes on High.
- Remove supernatant and wash pellet with 100 µl 70% ethanol. Spin for 1 minute, maximum speed.
- Remove as much of the ethanol as possible and dry tubes on bench for 5-10 minutes.
- Resuspend DNA in 40 µl of H2O containing 20 µg/ml RNase A.
- Determine DNA by spectrophotometry and test for proper plasmid recovery by restriction digest.
Solutions
Solution I
- 25mM Tris-HCl, pH 8.0
- 10mM EDTA
Solution II
- 0.2N NaOH
- 1.0% SDS
Solution III
- 3M Potassium Acetate, pH 4.8
3M Potassium Acetate, pH 4.8
- 60 ml 5M Potassium Acetate
- 11.5 ml Glacial Acetic Acid
- 28.5 ml H2O