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Barrington Laboratory Protocols
DNA Extraction
Barrington Lab Fern CTAB DNA extraction protocol for fresh, 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).
- Prepare CTAB buffer
Extraction buffer--pH 8.0 (makes 1L)
- 100 mM Tris-Hcl
- 280 mL of 5M NaCl
- 40 mL of 0.5 M EDTA
- 20 g of CTAB (Cetyltrimethyl ammonium bromide)
- Before starting extractions add per accession:
- 2.5 µL ß-mercaptoethanol
- 0.02 g PVP (polyvinylpryyolidone 360,000)
- Grind 0.1g fresh or 0.02g dried frond material with 500 µL extraction buffer and incubate @ 50-65ºC for 1 HR or OVERNIGHT for difficult herbarium accessions.
- Add 500 µL of 24:1 chloroform:isoamyl alcohol and vortex on a low setting for 1 minute.
- Centrifuge tubes for 10 minutes @ 15, 000 x g (max speed on the Eppendorf Centrifuge 5415C) and transfer aqueous layer to a fresh tube.
- The three layers you should have are: bottom – chloroform, middle - debris and proteins, top – aqueous phase
- Estimate the volume of the aqueous phase.
- Precipitate the DNA by adding 0.08 volumes of cold ammonium (or potassium) acetate and 0.54 volumes of cold isopropanol. Mix well.
- Let sit in freezer for 1HR or OVERNIGHT for difficult herbarium accessions.
- Recover DNA by centrifugation @15, 000 x g for 6 min.—save pellet and discard aqueous layer.
- Wash pellet with 700 µL 70% cold ethanol mix well and centrifuge at maximum speed for 3 minutes.
- Pour or pipette off the liquid, being careful not to lose the pellet with your DNA.
- Wash pellet with 700 µL 95% cold ethanol mix well and centrifuge at maximum speed for 3 minutes.
- Pour or pipette off the liquid, being careful not to lose the pellet with your DNA.
- Dry off excess ethanol in the vacuum dryer/speed vac for 20 minutes.
- Resuspend in ddH20 (50 µL). Allow to resuspend for 1 hour at 55ºC or overnight in the freezer.
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