The Polystichum Homepage

Welcome to the website for the hollyferns, members of the fern genus Polystichum and allies.
This website is maintained for the use of hollyfern enthusiasts and scholars
throughout the world by Dave Barrington at the University of Vermont in the United States.

Polystichum Homepage

Portrait of the Genus

Taxonomy

Species List

Diversity Map

Interactive Keys

Phylogeny

Cytology (PDF)

Barrington Web Page
(with publications)

Morphological Characters

Morphological Analysis

Lab Protocols

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).

  1. 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 L -mercaptoethanol
    • 0.02 g PVP (polyvinylpryyolidone 360,000)

  2. Grind 0.1g fresh or 0.02g dried frond material with 500 L extraction buffer and incubate @ 50-65C for 1 HR or OVERNIGHT for difficult herbarium accessions.
  3. Add 500 L of 24:1 chloroform:isoamyl alcohol and vortex on a low setting for 1 minute.
  4. 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

  5. Estimate the volume of the aqueous phase.
  6. Precipitate the DNA by adding 0.08 volumes of cold ammonium (or potassium) acetate and 0.54 volumes of cold isopropanol. Mix well.
  7. Let sit in freezer for 1HR or OVERNIGHT for difficult herbarium accessions.
  8. Recover DNA by centrifugation @15, 000 x g for 6 min.save pellet and discard aqueous layer.
  9. Wash pellet with 700 L 70% cold ethanol mix well and centrifuge at maximum speed for 3 minutes.
  10. Pour or pipette off the liquid, being careful not to lose the pellet with your DNA.
  11. Wash pellet with 700 L 95% cold ethanol mix well and centrifuge at maximum speed for 3 minutes.
  12. Pour or pipette off the liquid, being careful not to lose the pellet with your DNA.
  13. Dry off excess ethanol in the vacuum dryer/speed vac for 20 minutes.
  14. Resuspend in ddH20 (50 L). Allow to resuspend for 1 hour at 55C or overnight in the freezer.

Please be in touch with me with your questions about and insights into the holly ferns -