Materials
2X grinding buffer (ice-cold) 1% Sarkosyl solution
Lysis buffer (ice-cold) RNase A (10 mg/ml)
1% CTAB solution (65) 3 M sodium acetate (NaOAc)
5 M NaCl Isopropyl alcohol (ice-cold)
10 mM Tris, pH 8.0, 1 mM EDTA (TE 10, 1) EtOH (70% and 100%)
Labelled Oakridge & 30-ml Corex tubes (1 ea./sample) Weighing paper funnels
Phenol:chloroform:isoamyl alcohol (:CHCl3:IAA, Coffee grinder(s)
25:24:1)
For 8 samples:
1. Add sufficient vol. (190 l) -mercaptoethanol to (95 ml) 2X grinding buffer to achieve final concen. of 0.2%. Cover and store on ice.
2 Add sufficient vol. (65 l) -mercaptoethanol to (65 ml) lysis buffer to achieve final concen. of 0.1%. Cover and store on ice.
3. Excise ca. 2 g. fresh apical tissue (from each tree, one at a time), roll up tissue, grasp with large forceps, freeze in LN2 and grind in a pre-chilled (with dry ice) coffee grinder in the presence of dry ice (N.B. rinse single-edge razor with EtOH between plants and clean coffee grinder thoroughly between samples, to prevent cross-contamination).
4. Transfer ground tissue/dry ice to Oakridge tube with a funnel fashioned out of weighing paper.
5. After the dry ice has all sublimed away, add 10 ml ice-cold 2X grinding buffer (with -mercap-toethanol) and thoroughly grind tissue with the Polytron (N.B. I usually process eight samples at a time, in series, and grind each in sufficient dry ice so that after the eighth sample is done, there is little or no dry ice left in the first sample. In this way, I can proceed with this step right away. If sublimation is occurring too rapidly, it can be slowed by putting the tube on ice, but it is important to keep the samples cold).
6. Balance pairs of tubes using XS grinding buffer and centrifuge samples at 14,000 x g (in Sor-vall at 10,000 rpm in SA-600 rotor) for 10 min. at 4 C. Keep tubes on ice until they are centrifuged.
7. Pour off and discard the supernatant and resuspend the pellet in 7.5 ml ice-cold lysis buffer.
8. Add 1/10 vol. (750 l) of 10% Sarkosyl, shake vigorously and bring to room temperature.
9. Add 1/7 vol. (1.1 ml) 5 M NaCl and 3/10 vol. (2.25 ml) 1% CTAB solution, shake vigorously and incubate at 65 C for 20 min., shaking tubes occasionally.
10. Add equal vol. (11.6 ml) :CHCl3:IAA, balance pairs of tubes with XS :CHCl3:IAA, shake vigorously to form a emulsion, and spin in clinical tabletop at 3,000 rpm for 5 min. (room temperature).
11. Tranfer the aqueous phase to a 30-ml Corex tube with a Pasteur pipette, add 2/3 vol. (8 ml) isopropanol, balance pairs of tubes with isopropanol, cover tubes with Parafilm, mix by repeated inversion, and store on ice for 20-30 min. Spin at 14,000 x g (10,000 rpm in SA-600 rotor) for 10 min. at 4, pour off and discard supernatant. (N.B. the pellets can be covered and safely stored O.N. at -70 C, if necessary.)
12. Resuspend the pellet (by repeated up and down motion, using a yellow pipette tip from which the end has been removed) in 600 l TE (10, 1), add 5 l 10 mg/ml RNase A, and incubate at 37 C and 150 rpm for 4-5 hr.
13. Transfer sample to microfuge tube, add 1/10 vol. (60 l) of 3 M NaOAc, 700 l of :CHCl3:IAA, mix well.
14. Spin in microfuge at 14,000 x g for 5 min. and transfer whole aqueous phase to a new microfuge tube.
15. Repeat step 14 but only transfer ca. 500-600 l of aqueous to a new microfuge tube.
16. Add ca. 2 vol. (1 ml) 100% EtOH, chill samples at least 30 min. on ice (or store O.N. at -20 C) then pellet DNA at 14,000 x g for 5 min.
17. Aspirate supernatant, wash pellet with 500 l ice-cold 70% EtOH, aspirate all traces of EtOH, being careful not to dislodge or aspirate the pellet, and air-dry pellet for at least 15 min. (Be sure to rinse the needle in dH20 between samples to avoid cross-contamination.)
18. Add 200 l TE (10, 1) and store at 4 C until DNA dissolves. Remove tubes occasionally and gently flick to get DNA into solution faster. Do not vortex tubes or heat samples to facilitate dissolving process! Once DNA is completely dissolved, store at -20 C. Confirm that the DNA is intact by running a 2-l aliquot of each sample on a 0.8% agarose gel.
Solutions
2X Grinding Buffer (500 ml)
0.7 M sorbitol 64 g
100 mM Tris (pH 8.0) 50 ml of 1 M
10 mM EDTA (pH 8.0) 10 ml of 500 mM
0.2% spermine tetrachloride 1.0 g
0.2% spermidine trihydrochloride 1.0 g
2% polyvinylpyrrolidone (PVP) 10 g
10% PEG 4000 50 g
Lysis Buffer (250 ml)
0.35 M sorbitol 16 g
50 mM Tris (pH 8.0) 12.5 ml of 1 M
25 mM EDTA (pH 8.0) 12.5 ml of 500 mM
1% polyvinylpolypyrrolidone (PVPP) 2.5 g
1% CTAB solution (100 ml)
1% CTAB 1 g
0.7 M NaCl 14.3 ml of 5 M
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