JJSA Protocol for Silica-dried Leaves or Herbarium Samples: DNA Mini-preps
* Weigh or estimate a small amount of dried tissue (e.g., 10-50 mg of leaves)
* Warm buffer to 60°C. Add approximately 30 µl 2-mercaptoethanol to 15 ml 2X CTAB just before starting to grind.
* Measure out a small volume of sterilized sand and approximately equal amount of PVP into a mortar.
* Add tissue and 500 µl CTAB buffer to mortar and GRIND tissue thoroughly in mortar with pestle.
* Add 500 µl additional CTAB buffer (for a final volume of 1000 µl) and grind again. Carefully pour the ground material into a 1.5 ml microfuge tube.
* Incubate for 1 hr at 65-70°C.
* Add equal volume (ca. 700 µl) chloroform: isoamyl alcohol (24:1; will fill a 1.5 ml microfuge tube--holds 1.6 µl)
* Spin for 20 min until you have clear liquid above. (Hint: if viscous region is apparent just below leaf sample then you have removed from spin too early; i.e., from top to bottom may see clear region, viscous zone; leaf layer, dark zone)
* Transfer top aqueous phase to a new microfuge tube.
* Optional: repeat chloroform:isoamyl extraction if separation seems poor or if spin is inadequate. (I have had some trouble with microfuge tubes breaking on spins longer than 20 minutes)
* Add 540 µl cold isopropanol; mix & store overnight in - -20°C or 2-3 hrs @ -84°C
* Spin for 1-3 min at maximum speed.
* For samples with clear pellets, decant the supernatant & drain using a kimwipe. (For samples with gelatinous material above pellet; remove all but thin (mm or so) band of isopropanol with micropipettor, leaving gelatinous portion and pellet intact)
* Wash pellet with 500 µl 75% EtOH (note: some argue that DNA goes into water at 70%); invert tube several times (gelatinous material should dissolve)
* Spin for 3 min ; dry excess liquid with Kimwipe or on paper towel
* Remove supernatant; dry with kimwipe or on paper towel, leaving pellet intact.
* Dry with speedvac or in vacuum oven (20 min-1hr)
* Resuspend pellet in 80 µl 1X TE; gently pump pipettor to break up pellet.
* Add 8 µl of 5M 7.5 M ammonium acetate, and 180 µl of 100% EtOH
* Mix and place in -20°C freezer overnight or -80°C for 2 hr.
* Spin 2-3 min, decant supernatant (optional: add final wash of 75% EtOH & spin again)
* Decant supernatant, vacuum dry, and resuspend in 100 µl TE. Incubate at 37-40°C for 15-30 min to ensure DNA goes into solution.
* Verify presence of DNA using test gel. Aliquot and store at -84°C (long-term); -20°C (temporary) or 4°C (active use).
Procedure modified by S. Kephart via practice & discussions while in Andi Wolfe's lab; adapted in part from Doyle & Doyle (1987); K. Cullings (1992; Molecular Ecology); & D. Lockerman & R. Jansen (1996; in T. Stuessey & JS. Sohmer, Sampling the Green World)
J J= Jeff & Jane Doyle, Bob Jansen, Javier Franciso-Ortega, Jenny Xiang, S = Susan Kephart, Shannon Datwyler, Soltis Lab, A = Andi Wolfe
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