1. Harvest youngest unexpanded trifoliate leaves wherever possible.

2. Lyophilize or desiccate leaves until dry, typically 2-3 days. Dried leaves can be stored at -20 °C or -80°C for extended periods.

3. Load one or two trifoliates into individual wells of a 96 well square well deep-well plate. Add a BB to each well*, seal and pulverize tissue to a fine powder with a grinder.

4. Add 700ul fresh CTAB extraction buffer plus 0.1% beta-mercaptoenthanol to each well. Seal with adhesive cover and vortex very gently until well mixed.

5. Set the slurry in a 65°C water bath for 60' (or in the shaker/incubator for up to 2 hrs.). During incubation, vortex gently a couple of times to resuspend any tissue not in contact with the buffer. If using the shaker, set to 125 rpm. Remove and cool for several minutes at RT. Spin for 15' at 3600 rpm.

6. While CTAB plate(s) spin, set up two additional sets of racks/plates:

a. chloroform/isoamyl* step: in the hood, set up a 96 well rack of strip tubes and add 500 ul 24:1 chloroform/isoamyl alcohol to each tube.

b. isopropanol step: add 300 ul isopropanol to each well of a 96 well round well deep-well plate.

7. Transfer 400ul CTAB supernatant to each strip tube. Aspirate a couple of times til phases are completely mixed. Seal with adhesive cover.

8. Spin for 15' at 3600 rpm. After centrifugation, the upper (aqueous) phase should be clear.

9. Transfer the aqueous phase into 96 well round well isopropanol plates, mix by aspirating up and down a few times, then allow samples to sit at RT 15-30'.

10. Spin 15' at 3600 rpm. Carefully decant liquid, taking care not to dislodge pellets, which can be slippery. A rule of thumb is not to keep the plate inverted for more than a few seconds. Visually check that pellets remain at bottom of wells. Tamp plate on the bench a couple of times to reseat any pellets that may have moved up the walls of the plate.

11. Add 500 ul 70% ETOH and spin 15' at 3600 rpm. Following procedure in step 10 above, decant liquid, dry pellet thoroughly in shaker/incubator at 50°C (~30’) and re-suspend in 50-100 m l TE.

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*Tips:

Place a square well mat cap into a dish and pour BBs over it so that each dimple contains a single BB. Carefully align an inverted square well plate over the mat cap so that each dimple corresponds to a well. Flip plate quickly.

Because it’s difficult to see the aqueous/organic phase interface in the opaque 96 well plates, we use clear strip tubes for the chloroform step .

Add a pinch of Oil Red O powder to chloroform/isoamyl to aid in distinguishing the interface between the organic and aqueous phases.

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.