Fern CTAB DNA extraction protocol for both silica dried and herbarium quality material

This protocol contains elements of the extraction protocols of Doyle and Doyle (1986), Soltis Lab CTAB protocol on the web, and Dempster et al. (1999).

Extraction buffer--pH 8.0

100 mM Tris-Hcl

1.4 M NaCl

20 mM EDTA

2% (wt/vol) CTAB

0.4% (vol/vol) ß-mercaptoethanol

1% (wt/vol) PVP 360,000

1. Grind 0.1g fresh or 0.02g dried frond material with 500 µL extraction buffer and incubate @ 50-65 degrees C for 15-30 min. (keep samples on ice until they go into the hot water bath).

2. Add 500 µL of Sevag (24:1 chloroform:isoamyl alcohol) and shake tube for 10-20 min. (vortex on a low setting). Then centrifuge tubes for 10 minutes @ 15, 000 x g (max speed on the Eppendorf Centrifuge 5415C) and transfer aqueous layer (aka supernatant) to a fresh tube.

3. Precipitate the DNA by adding an equal volume of cold isopropanol and 0.5 volumes of 5 M NaCl to the supernatant and let sit at room temp. for 30 min.

4. Recover DNA by centrifugation @15, 000 x g for 20 min.—save pellet and discard aqueous layer.

5. Wash pellet with 700 µL 70% cold ethanol and centrifuge at max. speed for 5 min.

6. Suspend pellet in 180 µL TE (pH 8.0) and add 2 µL of 1mg/mL Rnase and incubate @ 37 degrees C for 30 min.

7. Add cold 7.5 M NH4Oac to reach a final concentration of 2.5 M (this is 60.6 µL)

8. Add two volumes of 95-100% ethanol and put in the freezer (-20 degrees C) overnight or in the deep freeze (-80 degrees C) for 1 hour.

9. Recover DNA by centrifuging tubes @ max speed for 20 min.

10. Wash pellet with 700 µL of 70% ethanol and centrifuge for 10 min.

11. Dry off excess ethanol in the vacuum dryer @ 60 degrees C for 5 min.

12. Resuspend in ddH20 (20-50 µL). Allow to resuspend for 1 hour at 55 degrees C or overnight in the fridge.