DNA Purification-Modified CTAB Procedure
August 25, 1993
This protocol originated in Murray and Thompson (1980) and then was modified by Richard Jorgensen and finally published in Wagner et. al 1987.
- Start with about 10 grams fresh needles.
- Chop into short pieces.
- Homogenize, with Polytron, in 100 ml "Ct Ext'n buffer" for less than 10 seconds, cold.
- Filter homogenate through several layers of cheesecloth and one layer of miracloth.
- Pellet organelles in the GSA rotor at 9000 RPM, for 15 minutes at 4oC.
- Resuspend pellet in 5 ml "Ct wash buffer", and 0.1% b-mercaptoethanol and bring to room temperature. Move to orange cap tubes.
- add 1/5 volume of 5% sarkosyl, set for 15 minutes at room temperature.
- add 1/7 volume 5M NaCl; mix gently by inversion.
- add 1/10 volume 8.6% CTAB, 0.7M NaCl.
- Incubate at 60oC for 15 minutes.
- add 10 ml chloroform/octanol (24:1), mix by inverting till an emulsion is formed about 20 times.
- Centrifuge at 5000 Xg for 10 minutes at room temperature. Use table top centrifuge.
- Transfer the upper aqueous phase to a 2nd tube (15 conical tube); add 2/3 volume isopropanol or EtOH and mix by inverting quickly 2 or 3 times to precipitate the DNA.
- Hook the precipitate out with a hooked pasteur pipet. Otherwise pellet the DNA at the lowest speed that will lightly compress the precipitate.
- Transfer the precipitate to a 50 ml polypropylene screwcap centrifuge tube containing about 20ml 76% EtOH/10mm NH4Ac; set for about 20 minute.
- Transfer precipitate to 1.5 ml microtube containing 1 ml of water.
- To completely dissolve the DNA, let it sit for 30 minutes at room temperature then gently disperse by inverting the tube several times. Incubate at 65oC if the DNA doesn't dissolve after this time.
- Store DNA at 4oC.
Ct Ext'n Buffer Ct Wash Buffer 50 mM Tris pH 8.0 50 mM Tris pH 8.0 5 mM EDTA 25 mM EDTA 0.35 M Sorbitol 0.35 M Sorbitol 0.1% BSA (Sigma A4503) (add first) (+0.1% b-mercaptoethanol) 0.1% b-mercaptoethanol (add just before using) 10% PEG 4000
- Murray MG, Thompson WF (1980) Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res 8:4321-4325.
- Wagner DB, Furnier GR, Saghai-Maroof MA, Williams SM, Dancik BP, Allard RW (1987) Chloroplast DNA polymorphisms in lodgepole and jack pines and their hybrids. Proc Natl Acad Sci USA 84:2097-2100.