Skip to Content

Plasmid Miniprep

  1. 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.
  2. Spin down 1.5 ml of overnight culture in Eppendorf for 1 minute on High.
  3. Asprirate supernatant and resuspend cell pellet in 100 µl Solution I.
  4. Add 200 µl Solution II mix gently by inversion 10-15 times.
  5. Add 150 µl Solution III, vortex briefly to mix and spin for 5 minutes on High.
  6. Transfer supernatant to fresh tube and add 500 µl phenol:chloroform, vortex and spin for 5 minutes on High.
  7. Transfer aqueous layer to fresh tube and add 1 ml ethanol, mix well by inversion and spin for 5 minutes on High.
  8. Remove supernatant and wash pellet with 100 µl 70% ethanol. Spin for 1 minute, maximum speed.
  9. Remove as much of the ethanol as possible and dry tubes on bench for 5-10 minutes.
  10. Resuspend DNA in 40 µl of H2O containing 20 µg/ml RNase A.
  11. 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

© 2014 The University of Texas MD Anderson Cancer Center