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single leaf DNA prep

Single (or two) leaf genomic DNA extraction
Arabidopsis DNA Prep Based on the Dellaporta Prep
(provided to us by Carolyn Napoli, University of Arizona; 5/3/01)

1. Collect 50 mg of fresh leaves (1-2 leaves) in a 1.5 ml eppendorf tube and place at -80C. Pre-chill a metal heat block at -80C. Place tubes containing frozen leaves in the pre-chilled metal block. Place chilled metal block in a small styrofoam container (provides minimal insulation). We do 18-20 tubes at one time, then pre-chill the metal block before grinding another round of tissue.

2. Open tubes and place a blue pestle in each tube. Grind tissue, leave pestle in the tube and remove the tube from the block. Add 480 ul extraction (lysis) buffer and grind tissue some more. Add 37.5 ul of 20% SDS and mix by inverting the tubes 5-8 times.

3. Heat samples at 65C for 10 minutes. Add 94 ul 5M potassium acetate and invert tubes to mix. Place on ice for 5 minutes.

4. Spin tubes for 5 minutes. While tubes are spinning, prepare Phase Lock Gel tubes (Eppendorf, Heavy, 2 ml) by spinning them at 12,000 RPM for 2 minutes to pellet the gel glob. Transfer the supernatant (~600 ul) to a phase lock tube (can pour the supernatant, don't worry about floating leaf pieces). Add 600 ul phenol-chloroform and mix by inversion. (Caution: do not vortex Phase Lock tubes)

5. Spin tubes for 5 minutes at 12,000 RPM, pour off supernatant into a clean eppendorf tube.

6. Add 360 ul (0.6 volume) isopropanol, mix and centrifuge for 3 minutes.

7. Rinse pellet with 70% EtOH and then rinse with 95% EtOH (may have to spin again). Air-dry and resuspend in 30 ul TE. Use 10-15 ml for a Southern blot.

Lysis Buffer
100 mM Tris pH 8; 50 mM EDTA; 500 mM NaCl