1. Harvest young barely expanded trifoliate leaves or the first unifoliate leaves.
2. Lyophilize leaves for 1 to 2 days. Lyophilized leaves can be stored at -20°C for extended periods.
3. Load one or two small leaves into individual wells of a 96 well deep-well rack or 1.5 ml eppendorf tubes. Add a BB to each well and grind to a fine powder with grinder. Add 700ml fresh extraction buffer (see recipe). Cover with adhesive cover and vortex gently until well mixed.
4. Set the slurry in a 65°C water bath for 30' (or in the shaker/incubator for up to 2 hrs.). During incubation vortex gently a couple of times. Remove and allow to cool for several minutes at room temperature. Spin for 15' at 3600 rpm.
5. Transfer the supernatant to new 1.5 ml tubes or rack (if you like, you can add 2ml RNase at this stage, but that's optional. Mix gently and incubate at 37°C for 30-60').
6. Add 500ml 24:1 chloroform/isoamyl alcohol and aspirate gently until phases are thoroughly mixed.
7. Spin at 3600 rpm for 15'. After centrifugation, the upper (aqueous) phase should be relatively clear.
8. Aspirate aqueous phase into new tubes or rack, taking care not to disturb the underlying organic phase. Add 300ml isopropanol and mix gently by inversion (don't vortex). Allow to sit at room temperature for 15-30'.
9. Spin at 3600 rpm for 15'. Decant liquid.
10. Add 500ml 70% ETOH and spin at 3600 rpm for 15', decant liquid or vacuum dry (dry in shaker/incubator at 70°C for 30'), and re-suspend in 50-100ml dH₂O.

CTAB extraction buffer		Final concentration
10g CTAB		2%
140ml 5 M NaCl		1.4 mM
25ml 2 M Tris-Cl pH 8.0		100mM
20ml 0.5 M EDTA		20mM
adjust to 500ml with Type 1 H2O

Reference
Keim, et al. Soybean Genetics Newsletter, 1988.