Alkaline Lysis Plasmid Miniprep
Based on Maniatis and Short Protocols in Molecular Biology edited by Ausubel, et al.
- Inoculate 2 ml 2X YT media (supplemented with the appropriate antibiotic) with a medium sized bacterial colony. Incubate at 37°C overnight.
- 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.
- 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
- Let sit 5 minutes at room temperature.
- 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 —
- 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°.
- Spin for 5 minutes in a microcentrifuge to pellet the cell debris and chromosomal DNA. Repeat if necessary.
- 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.
- 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.
- Add two volumes 95% ethanol. Incubate for at least 20 minutes at -20°C.
- Spin in the microcentrifuge for 10 minutes. Decant the supernatant and wash the pellet once with 70% ethanol.
- Dry the pellet in the SpeedVac for 10 minutes.
- Resuspend the pellet in 30 µl TE.
- 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.