Department of Ecology, Evolution and Organismal Biology
Iowa State University, Ames, IA 50011, USA
Our lab is currently using Qiagen's Plant DNeasy extraction kit for most DNA extractions. We have tested that kit against several other methods (including the one described below), and we found it to be the simplest method that gives high-quality, clean DNA (note). However, we also have used a modified CTAB DNA (described below) isolation protocol that is suitable for cotton and other plants with abundant polysaccharides & polyphenolics.
This protocol is modified from the procedure reported by Tel-Zur, N. et al. in Plant Biology Reporter (1999, vol. 17, pp. 249-54). It has produced high-quality DNA from various malvaceous species, including Gossypioides kirkii, a very recalcitrant species for DNA, and from old cotton fibers. The extraction buffer is responsible for getting rid of many cell components but retains the organelles; nuclei are also burst open upon its introduction. The high-salt CTAB buffer specifically separates DNA from other substances, such as proteins. Sarkosyl is a detergent and thus disrupts membranes. Phenol and chloroform are both protein removers.
Reagents and Solutions
- Extraction Buffer: 100 mM Tris-HCl at pH 8.0, containing 0.35M sorbitol, 5mM EDTA at pH 8.0, and 1% 2-mercaptoethanol (added just before us) (keep entire solution ice cold)
- High-Salt CTAB Buffer: 50 mM Tris-HCl at pH 8.0, containing 4M NaCl, 1.8% CTAB (mixed alkyltrimethylammonium bromide), and 25 mM EDTA at pH 8.0
- Sarkosyl (30% aqueous solution)
- Chloroform: isoamyl alcohol (24:1)
- Isopropanol (100%)
- TE buffer, 1´ solution
- RNase A (10 mg/mL)
- Sodium acetate (3M, at pH 5.2)
- Phenol: chloroform (1:1; make just before use)
- Ethanol (both 70% and 100%, both cold)
1. Grind 1-2 g of fresh tissue with mortar and pestle in liquid nitrogen.
2. Transfer the powder into 50 mL tubes and add 20 mL ice-cold extraction buffer. Gently mix and/or invert tubes for about 5 minutes.
3. Centrifuge the tubes at 4°C, 5000g (~6200 rpm) for 10 minutes. [We use an IEC Centra MP4R tabletop centrifuge fitted with rotor model 801 (6-place fixed-angle 45°)].
4. Decant the supernatant.
5. Add another 20 mL of ice cold extraction buffer and wash for approximately 5 minutes as before.
6. Centrifuge as in step #3.
7. Decant the supernatant.
8. Add 5 mL ice cold extraction buffer and gently mix briefly as before.
9. Add 3.5 mL of the high-salt CTAB buffer and 0.3 mL 30% sarkosyl. Incubate in a shaker (50-60 rpm) for 1 to 1 ½ hours.
10. Extract with chloroform. [Note: Do this by adding an equal volume of chloroform and gently mixing. Shake gently, and then centrifuge at 5000g for 10 minutes. You will note the formation of three layers: an upper phase (aqueous phase), a thin interphase, and a lower phase. The phase you want to collect is the upper phase, which contains your DNA. The interphase contains remnants of the cells and various junk. The lower phase will be mostly chloroform, proteins, and a composite of biochemicals. When pipetting out the upper phase, don’t get greedy – it’s especially important not to pick up any of the inter- or lower phases. It’s better to leave some of the upper phase behind than trying to get every last drop and risk picking up some of the other layers and contaminating your sample.]
11. Add 2/3 (v/v) cold isopropanol. Invert several times.(*This is a potential stopping-point. After adding the isopropanol, store at 4°C overnight or during daytime break.)
12. Centrifuge at 5000g for 10 minutes.
13. Decant the solution, leaving the pellet behind. Add mL cold 70% ethanol and swirl to mix components. Centrifuge at 5000g for 5 minutes.
14. Decant the solution, leaving the pellet behind. Dry the pellet for 30 minutes in a fume hood or upside down on paper towel at the bench.
15. Add 500 uL of 1´ TE buffer and dissolve pellet in water bath (up to 60°C). Transfer fluids to 1.7 mL microcentrifuge tubes.
16. Add 5-10 uL RNase A and incubate at room temperature or 37°C for 30-60 minutes.
17. Add 500 uL phenol, thoroughly mix by inversion or gentle vortexing, and centrifuge at 10,000g (~12,400 rpm) for 10 minutes. [In this and the remaining centrifugation steps we use an Eppendorf 5417c centrifuge with an F45-30-11 rotor. The maximum speed of this model is 20, 817 rpm.] Extract the upper (aqueous) phase and transfer to a new set of microcentrifuge tubes. Use the same caution as in step #10.
18. Add 500 uL phenol:chloroform (1:1; make just before use), thoroughly mix by inversion or gentle vortexing, and centrifuge at 10,000g (~12,400 rpm) for 10 minutes. Extract the upper (aqueous) phase and transfer to a new set of microcentrifuge tubes. Use the same caution as in step #10.
19. Add 500 uL chloroform, thoroughly mix by inversion or gentle vortexing, and centrifuge at 10,000g (~12,400 rpm) for 10 minutes. Extract the upper (aqueous) phase and transfer to a new set of microcentrifuge tubes. Be extremely careful not to pipette up any of the inter- or lower phase at this specific extraction!
20. Add 2 volumes of ice cold 100% ethanol combined with 1/10 volume sodium acetate. Store at -20°C for 30 minutes.
21. Centrifuge the tubes at the highest speed for 15 minutes. This will pellet the DNA.
22. Add 300 uL cold 70% ethanol and let tube sit at room temperature for 30-60 minutes.
23. Centrifuge at 5000g for 5 minutes.
24. Dry the pellet. [We use a vacuum centrifuge (speed-vac) with or without heat on.]
25. Dissolve the pellet in either water or TE (depending on size, about 200-300uL of solvent). [If you expect low yield, only use 50-100 uL of solvent.]
Protocol updated December 2001. Many thanks to all those who contributed.