Bacterial Minipreps

  1. Select a single bacterial colony and grow at 37oC overnight in 5 ml LB containing 50 ug/ml ampicillin.

  2. Harvest 1.5 ml of culture by microcentrigugation at 8,000 rpm for 2 minutes.

  3. Aspirate off the supernatant and resuspend the cell pellet in 100 ul P1 buffer.

  4. Add 100 ul P2 buffer and gently invert 4-6 times to mix thoroughly. Do not vortex.

  5. Add 100 ul P3 buffer and gently invert 4-6 times to mix thoroughly. Do not vortex.

  6. Pellet cellular debris by microcentrifugation at 14,000 rpm for 10 minutes.

  7. Withdraw the supernatant to a new 1.5 ml eppendorf tube and add 240 ul isopropanol. Gently invert 4-6 times to mix thoroughly. Incubate 2 minutes at room temperature.

  8. Pellet plasmid DNA by microcentrifugation at 14,000 rpm for 10 minutes.

  9. Aspirate supernatant from pellet.

  10. Pulse-spin the DNA to collect the remaining supernatant on the sides of the tube.

  11. Aspirate supernatant from pellet.

  12. Resuspend plasmid DNA in 50 ul TE or distilled water.


P1 BUFFER
(20 mM Tris-HCL, pH 8.0, 1 mM EDTA, 1 ug/ml RNAse)

TO MAKE 500 ml:

  • 10 ml 1M Tris, pH 8 stock solution
  • 1.0 ml 0.5 M EDTA stock solution
  • 500 ul 10 mg/ml RNAse stock solution

P2 BUFFER
(200 mM NaOH, 1% SDS)

TO MAKE 500 ml:

  • 10 ml 10 M NaOH stock solution
  • 50 ml 10% SDS stock solution

P3 BUFFER
(3 M KOAc, pH 5.5)

TO MAKE 500 ml:

  • 147.6 g KOAc
  • bring pH to 5.5 using acetic acid (~55 ml)