DNA Miniprep using CTAB
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! It is a great protocol to extract DNA from plants.
- Add 1% beta-mercaptoethanol to the CTAB-buffer
- Preheat the CTAB-buffer to 65 °C
- Prepare 600 µl CTAB-buffer in an eppi
- Grind tissue into a fine powder
- Transfer tissue-powder to the eppi and mix very well
- Incubate at 65 °C for 30 min
- Add 600 µl chloroform and mix very well
- Spin down at max speed for 2 min
- Transfer upper phase into a new eppi
- Add 600 µl chloroform and mix well
- Spin down at max speed for 2 min
- Transfer upper phase into a new eppi
- Add 450 µl of isopropanol and mix well
- Optional: Store for 30 min at -20 °C
- Spin down at max speed for 15 min
- Keep the pellet, discard supernatant
- Add 600 µl 70% ethanol to wash and mix well
- Spin down at max speed for 5 min
- Keep the pellet, discard supernatant
- Dissolve pellet in 90 µl water for 15 min at 65 °C
- Spin down at max speed for 5 min
- Transfer the liquid into a new eppi
This contanis RNA and DNA. If you prefer to use RNAse to get rid of RNA:
Add 1 µl RNAse solution and incubate 30 min at 37 °C.
If you do not want to purify further go directly to step 34.
Most protocols end here! - Add 30 µl 8 M LiCl and mix well
- Precipitate the RNA for at least 30 min at -20 °C
- Spin down at max speed for 30 min at 4 °C
- Keep the supernatant, discard pellet
- Add 80 µl isopropanol and mix well
- Spin down at max speed for 20 min
- Keep the pellet, discard supernatant
- Add 100 µl 70% ethanol to wash and mix well
- Spin down at max speed for 5 min
- Keep the pellet, discard supernatant
- Dissolve pellet in 100 µl water for 15 min at 65 °C
- Store DNA at -20 °C for long term storage
Buffers and Solutions
CTAB Buffer
| 100 ml | 500 ml | ||
| CTAB (Cetyl Trimethyl Ammonium Bromide) | 2% | 2 g | 10 g |
| NaCl | 1.4 M | 8.1 g | 40.6 g |
| 0.5 M EDTA, pH 8.0 | 20 mM | 4 ml | 20 ml |
| 1 M TRIS, pH 8.0 | 100 mM | 10 ml | 50 ml |
| Polyvinylpyrolidone (PVP40) (M.W. 40,000) | 2% | 2 g | 10 g |
| water | add to 100 ml | add to 500 ml |
The buffer takes some time to dissolve. Some people like to
autoclave it before use (me), others prefer to make it fresh.
Before use add 1 % beta-mercaptoethanol!
Commented Protocol:
1. Add 1% beta-mercaptoethanol to the CTAB-buffer
The buffer is not stable, use it within one day.
2. Preheat the CTAB-buffer to 65 °C
The buffer is not stable, use it within one day.
3. Prepare 600 µl CTAB-buffer in an eppi
Directly go to the next step after each sample.
4. Grind tissue into a fine powder
This is best done in a mortar for big amounts with liquid nitrogen or in a cap-shaker with glass beats for smaller amounts. If a cap shaker is used, 600 µl CTAB-buffer can be added directly following shaking.
5. Transfer tissue-powder to the eppi and mix very well
Do not take too much or too few. Use not more than 5 mg of dried tissue for extraction. 75 mg fresh poplar leaves works also well (just to give some guidance values). If you take too much, the lysis will not work, if you take too few, the DNA will not precipitate efficient enough.
6. Incubate at 65 °C for 30 min
Shake from time to time. Some protocols only incubate for 10 min. Probably even shorter times are sufficient. If you are in a hurry try first if shortening time decreases yield. I extended already up to 1.5 hours without decreasing the yield (due to many samples).
7. Add 600 µl chloroform and mix very well
After this step I wait till I processed all samples.
8. Spin down at max speed for 2 min
Handle the eppis carefully to not disturb the interphase.
9. Transfer upper phase into a new eppi
It is no problem if you take a little chloroform. But do not take anything of the interphase.
10. Add 600 µl chloroform and mix well
11. Spin down at max speed for 2 min
This time no white interface should form.
12. Transfer upper phase into a new eppi
Try to take none of the chloroform at this step.
13. Add 450 µl of isopropanol and mix well
This precipitates all nucleic acids. Some protocols use 600 µl.
14. Optional: Store for 30 min at -20 °C
This may increase yield, but also more RNA may precipitate.
15. Spin down at max speed for 15 min
Probably 10 min are sufficient.
16. Keep the pellet, discard supernatant
The pellet should be white and easily visible.
17. Add 600 µl 70% ethanol to wash and mix well
This washes off the salts.
18. Spin down at max speed for 5 min
Probably even 1 min would be sufficient.
19. Keep the pellet, discard supernatant
Do not dry the pellet at this step.
20. Dissolve pellet in 90 µl water for 15 min at 65 °C
21. Spin down at max speed for 5 min
Probably even 1 min would be sufficient.
22. Transfer the liquid into a new eppi
This contanis RNA and DNA. If you prefer to use RNAse to get rid of RNA:
Add 1 µl RNAse solution and incubate 30 min at 37 °C.
If you do not want to purify further go directly to step 34.
Most protocols end here!
23. Add 30 µl 8 M LiCl and mix well
This will precipitate the RNA only.
24. Precipitate the RNA for at least 30 min at -20 °C
25. Spin down at max speed for 30 min at 4 °C
26. Keep the supernatant, discard pellet
The RNA pellet might be transparent and hardly visible.
27. Add 80 µl isopropanol and mix well
To precipitate the DNA.
28. Spin down at max speed for 20 min
29. Keep the pellet, discard supernatant
30. Add 100 µl 70% ethanol to wash and mix well
This washes off the salts.
31. Spin down at max speed for 5 min
Probably even 1 min would be sufficient.
32. Keep the pellet, discard supernatant
Do not dry the pellet at this step.
33. Dissolve pellet in 100 µl water for 15 min at 65 °C
Most of the time pipetting up and down is sufficient.
34. Store DNA at -20 °C for long term storage
Known Issues:
- This protocol is very common and has been modified so many times. Many other protocols only incubate for 10 min at 65 °C and make only one chloroform extraction. It can probably be further adapted, this protocol aims to succeed and provide high yields.
- Works well on Tobacco, Tomato and Arabidopsis.
- If you don't get DNA with this protocol, search for protocols specific for your plant and be prepared for difficulties.
References and Comments:
This is a basic protocol used in the plant field to extract DNA from all kinds of tissues. It is popular because it works really well. I did it several times and the DNA was always great.
How to cite this page in publications:
This document can be cited like this:
Untergasser A. “DNA Miniprep using CTAB”
Untergasser's Lab. Summer 2008. (include here
the date when you accessed these page).
<http://www.untergasser.de/lab/protocols/miniprep_dna_ctab_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.