from Medicago truncatula

(Cook lab-U.C. Davis; drcook@ucdavis.edu)

Protocol for 1g of fresh tissue

1. Preheat 6 ml of CTAB isolation buffer in a tube with a cap to 65°C in a water bath.

CTAB buffer: 2% (w/v) CTAB
1.4M NaCl
0.2% (v/v) b-mercaptoethanol
20mM EDTA (use 0.5M pH 8.0 stock)
100mM Tris-HCl, pH 8.0

(For plants with a high concentration of phenolic compounds, 1% (w/v) of polyvinylpyrollidone (PVP-40) can be added to the isolation buffer).

(For plants containing high polysaccharide levels, the percentage of CTAB can be increased to 3% w/v)

2. Grind 1g of fresh (or frozen at -80°C) tissue in liquid nitrogen to a powder and transfer it quickly to the preheated CTAB buffer. (Do not let the powder thaw, as this will result in degraded DNA).

3. Swirl gently to mix and incubate at 60°C for 30 min.

4. Extract once with an equal volume of phenol:chloroform:isoamyl-alcohol (25:24:1), mixing gently. Centrifuge to separate the phases.

5. Recover the upper phase. Add RNase (DNase-free) to a final concentration of 20µg/ml and incubate at 37°C x 15 minutes.

6. Repeat the phenol:chloroform:isoamyl-alcohol extraction and centrifugation until the aqueous interphase is clear.

7. Extract the aqueous phase once with an equal volume of chloroform. Recover the aqueous phase following centrifugation.

8. Precipitate the DNA by adding 1/10 vol NaOAc (0.3M stock, pH 5.2) and 0.6 vol isopropanol. Hold at -20°C for 1 hr.

9. Pellet the DNA by centrifugation. Wash pellet once with 75% ethanol.

10. Centrifuge to solidify pellet. Remove ethanol and resuspend in TE buffer (10mM Tris/1mM EDTA, pH 8.0).

[Method modified from Focus (1990) 12(1)].

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