Protocols: Miniprep DNA extractions

JJSA Protocol for Silica-dried Leaves or Herbarium Samples: DNA Mini-preps


SAMPLE PREPARATION & GRINDING

* Weigh or estimate a small amount of dried tissue (e.g., 10-50 mg of leaves)

* Warm buffer to 60C. Add approximately 30 l 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 l CTAB buffer to mortar and GRIND tissue thoroughly in mortar with pestle.

* Add 500 l additional CTAB buffer (for a final volume of 1000 l) and grind again. Carefully pour the ground material into a 1.5 ml microfuge tube.

EXTRACTION AND ISOLATION OF DNA

* Incubate for 1 hr at 65-70C.

* Add equal volume (ca. 700 l) chloroform: isoamyl alcohol (24:1; will fill a 1.5 ml microfuge tube--holds 1.6 l)

* 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)

DNA PRECIPITATION

* Add 540 l cold isopropanol; mix & store overnight in - -20C or 2-3 hrs @ -84C

* 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 l 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)

FINAL CLEANING & RESUSPENSION OF DNA

* Resuspend pellet in 80 l 1X TE; gently pump pipettor to break up pellet.

* Add 8 l of 5M 7.5 M ammonium acetate, and 180 l of 100% EtOH

* Mix and place in -20C freezer overnight or -80C 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 l TE. Incubate at 37-40C for 15-30 min to ensure DNA goes into solution.

* Verify presence of DNA using test gel. Aliquot and store at -84C (long-term); -20C (temporary) or 4C (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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Please send your suggestions, comments, corrections to wolfe.205@osu.edu
Last updated August 19, 1998.