DNA-Miniprep - Quick & Dirty Alkaline Lysis

by A. Untergasser (contact address and download at www.untergasser.de/lab)
Version: 1.0 - Print Version (.PDF)

ATTENTION: This is a low priced protocol. Use it preferably!
People like it because it's fast and you can pour over the liquids (no need to pipett). Be aware that the DNA is not very clean. It works for digests, but more tricky things may get difficult.

  1. Pick colony and grow in 3 ml LB over night at 37°C
  2. Spin down 1.5 ml for 20 sec at max speed; store the rest at 4°C
  3. Pour off supernatant leaving about 50 - 100 µl
  4. Resuspend cells by vortexing
  5. Add 300 µl TENS (0.1M NaOH/0.5% SDS in TE)
  6. Mix by inverting about 5 times
  7. Add 150 µl of 3M NaAc pH5.2
  8. Mix by inverting about 5 times
  9. Spin down for 5 min at max speed
  10. Transfer the supernatant to new eppi
    Here you can pour over, no need to pipett.
  11. Add 900 µl of 96% ethanol
  12. Mix by vortexing for a few secs
  13. Spin down for 5 min at max speed
  14. Pour off and discard supernatant
  15. Add 500 µl 70% ethanol
  16. Mix by vortexing for a few secs
  17. Pour off all supernatant
  18. Spin for 30 seconds
  19. Remove all remaining supernatant by pipetting it off
  20. Leave the tubes open for at least 10 minutes to evaporate all ethanol
  21. Dissolve the dried pellet in 30 µl water containing 20 µg/ml RNase
  22. Vortex until a milky suspension is formed
  23. Spin down the insoluble white stuff for 2 min at max speed
  24. Use the clear supernatant in subsequent digestions

Commented Protocol:

1. Pick colony and grow in 3 ml LB over night at 37°C

3 ml LB are fine if you prep 1.5 ml, use 4 ml if you want to prep 3 ml. Then you have still enough to inoculate a maxiprep, 500 µl for a glycerol stock or similar things...

2. Spin down 1.5 ml for 20 sec at max speed; store the rest at 4°C

Here we spin very short so the pellet is not too compact. Therefore resuspeding by vortexing will still work.

3. Pour off supernatant leaving about 50 - 100 µl

We reuse the medium to resuspend the cells. This does not have to be acurate.

4. Resuspend cells by vortexing

The cells should resuspend within 15 - 30 sec. If it takes longer, reduce the time for centrifugation.

5. Add 300 µl TENS (0.1M NaOH/0.5% SDS in TE)

This will start the lysis of the bacteria.

6. Mix by inverting about 5 times

7. Add 150 µl of 3M NaAc pH5.2

8. Mix by inverting about 5 times

9. Spin down for 5 min at max speed

It will pellet the debris.

10. Transfer the supernatant to new eppi

Here you can pour over, no need to pipett.

11. Add 900 µl of 96% ethanol

This will precipitate the DNA.

12. Mix by vortexing for a few secs

13. Spin down for 5 min at max speed

It will pellet the DNA.

14. Pour off and discard supernatant

Keep an eye on the pellet to avoid losing it.

15. Add 500 µl 70% ethanol

16. Mix by vortexing for a few secs

Don't vortex too much, the pellet should still be intact at the end.

17. Pour off all supernatant

18. Spin for 30 seconds

19. Remove all remaining supernatant by pipetting it off

This is still the fastest way to remove it completely.

20. Leave the tubes open for at least 10 minutes to evaporate all ethanol

Ethanol can interfere with enzymatic reactions.

21. Dissolve the dried pellet in 30 µl water containing 20 µg/ml RNase

Don't forget the RNAse or you will have huge amounts of RNA on your gel.

22. Vortex until a milky suspension is formed

23. Spin down the insoluble white stuff for 2 min at max speed

24. Use the clear supernatant in subsequent digestions

This DNA solution is sufficient for digestions.

Known Issues:

References and Comments:

I got this protocol from Jan Verver. He likes it because it's fast and simple. I never did it, because I prefer the rapid boiling method which results in cleaner DNA.

How to cite this page in publications:

This document can be cited like this:

Verver J. and Untergasser A. “DNA-Miniprep - Quick & Dirty Alkaline Lysis” Untergasser's Lab. Summer 2008. (include here the date when you accessed these page).
<http://www.untergasser.de/lab/protocols/miniprep_quick_alkaline_lysis_v1_0.htm>.

Please Do Not Reprint This Article:

This article is copyrighted. Please do not reproduce this article in whole or part, in any form, without obtaining my written permission.