Miniprep

Alkaline Lysis Plasmid Miniprep

Based on Maniatis and Short Protocols in Molecular Biology edited by Ausubel, et al.

Protocol

  1. Inoculate 2 ml 2X YT media (supplemented with the appropriate antibiotic) with a medium sized bacterial colony. Incubate at 37°C overnight.
  2. Transfer 1.5 ml of the bacterial suspension to a 1.5 Epindorf tube. Spin for 20 seconds in a microfuge. Remove the supernatant using a P200.
  3. Resuspend the pellet in 110 µl of Solution I. Complete dispersion of the pellet is critical.
    Solution I
    [Stock] Reagent Amount [Final]
    2 M Glucose 2.5 ml 50 mM
    1 M Tris-HCl (pH = 8) 2.5 ml 25 mM
    0.5 M EDTA (pH = 8) 2 ml 10 mM
    — H2O 93 ml —
    Autoclave
  4. Let sit 5 minutes at room temperature.
  5. Add 200 µl of Solution II. Mix gently and incubate on ice for 10 minutes.
    Solution II
    [Stock] Reagent Amount [Final]
    10 N NaOH 0.2 ml 200 mN
    20 % SDS 0.5 ml 1 %
    — H2O 9.3 ml —
  6. Add 150µl ice-cold Solution III. Vortex at the highest setting for 2 seconds to mix. Place on ice for at least 5 minutes.
    Solution III
    Reagent Amount
    KOAc 29.44 g
    Glacial Acetic Acid 11.5 ml
    H2O to 100 ml
    Dissolve the KOAc in ~50 ml H2O, add the acetic acid
    then add H2O to reach 100 ml. Store at -20°.
  7. Spin for 5 minutes in a microcentrifuge to pellet the cell debris and chromosomal DNA. Repeat if necessary.
  8. Transfer the supernatant to a new 1.5 ml Epindorf tube. Add 450 µl phenol. Vortex 20 seconds to 1 minute. Spin in the microcentrifuge for 10 minutes.
  9. Transfer the aqueous (top) phase to a new 1.5 ml Epindorf tube. Extract with an equal volume of chloroform:isoamyl alcohol (24:1). Spin for 5 minutes in the microcentrifuge. Transfer the aqueous (top) phase to a new 1.5 ml Epindorf tube.
  10. Add two volumes 95% ethanol. Incubate for at least 20 minutes at -20°C.
  11. Spin in the microcentrifuge for 10 minutes. Decant the supernatant and wash the pellet once with 70% ethanol.
  12. Dry the pellet in the SpeedVac for 10 minutes.
  13. Resuspend the pellet in 30 µl TE.
  14. In digestions use 1 µl of the plasmid prep per reaction.

This page is, as always, Copyright (c) 1998 by Craig D. Amundsen. All rights reserved.