Protocol

Preparation of Plasmid DNA by Alkaline Lysis with SDS: Minipreparation

This protocol was adapted from Molecular Cloning, 3rd edition, by Joseph Sambrook and David W. Russell. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2001

INTRODUCTION

Plasmid DNA is isolated from small-scale (1-2 ml) bacterial cultures by treatment with alkali and SDS.

MATERIALS

Alkaline lysis solution I

For preparations of plasmid DNA that are to be subjected to further purification by chromatography (please see Purification of Plasmid DNA by Chromatography), sterile Alkaline lysis solution I may be supplemented just before use with the appropriate volume of 20 mg/ml DNase-free RNase A (pancreatic RNase) to give a final concentration of 100 µg/ml

Alkaline lysis solution II

Alkaline lysis solution III

Antibiotic for plasmid selection

Ethanol

Optional, please see Step 5.

Phenol:chloroform (1:1, v/v)

Rich medium

STE

TE (pH 8.0) containing 20 µg/ml RNase A

METHOD

1. Inoculate 2 ml of rich medium (LB, YT, or Terrific Broth) containing the appropriate antibiotic with a single colony of transformed bacteria. Incubate the culture overnight at 37°C with vigorous shaking.

2. Pour 1.5 ml of the culture into a microcentrifuge tube. Centrifuge at maximum speed for 30 seconds at 4°C in a microcentrifuge. Store the unused portion of the original culture at 4°C.

3. Remove the medium by aspiration, leaving the bacterial pellet as dry as possible.

4. Resuspend the bacterial pellet in 100 µl of ice-cold Alkaline lysis solution I by vigorous vortexing.

5. Add 200 µl of freshly prepared Alkaline lysis solution II to each bacterial suspension. Close the tube tightly, and mix the contents by inverting the tube rapidly five times. Do not vortex! Store the tube on ice.

6. Add 150 µl of ice-cold Alkaline lysis solution III. Close the tube and disperse Alkaline lysis solution III through the viscous bacterial lysate by inverting the tube several times. Store the tube on ice for 3-5 minutes.

7. Centrifuge the bacterial lysate at maximum speed for 5 minutes at 4°C in a microcentrifuge. Transfer the supernatant to a fresh tube.

8. (Optional) Add an equal volume of phenol:chloroform. Mix the organic and aqueous phases by vortexing and then centrifuge the emulsion at maximum speed for 2 minutes at 4°C in a microcentrifuge. Transfer the aqueous upper layer to a fresh tube.

9. Precipitate nucleic acids from the supernatant by adding 2 volumes of ethanol at room temperature. Mix the solution by vortexing and then allow the mixture to stand for 2 minutes at room temperature.

10. Collect the precipitated nucleic acids by centrifugation at maximum speed for 5 minutes at 4°C in a microcentrifuge.

11. Remove the supernatant by gentle aspiration as described in Step 3 above. Stand the tube in an inverted position on a paper towel to allow all of the fluid to drain away. Use a Kimwipe or disposable pipette tip to remove any drops of fluid adhering to the walls of the tube.

12. Add 1 ml of 70% ethanol to the pellet and invert the closed tube several times. Recover the DNA by centrifugation at maximum speed for 2 minutes at 4°C in a microcentrifuge.

13. Remove all of the supernatant by gentle aspiration as described in Step 3.Take care with this step, as the pellet sometimes does not adhere tightly to the tube.

14. Remove any beads of ethanol that form on the sides of the tube. Store the open tube at room temperature until the ethanol has evaporated and no fluid is visible in the tube (5-10 minutes).

15. Dissolve the nucleic acids in 50 µl of TE (pH 8.0) containing 20 µg/ml DNase-free RNase A (pancreatic RNase). Vortex the solution gently for a few seconds. Store the DNA solution at -20°C.

REFERENCES

1. Ish-Horowicz, D. and Burke, J.F. 1981. Rapid and efficient cosmid cloning. Nucleic Acids Res. 9: 2989–2998.[Abstract/Free Full Text]

2. Birnboim, H.C. and Doly, J. 1979. A rapid alkaline procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7: 1513–1523.[Abstract/Free Full Text]