Mollusc DNA Extraction - CTAB

Going with my theory that information is better cataloged on the internet than sitting collecting virtual dust on my hard drive, I'm posting some protocols in response to shortysquab's request for mollusc DNA extraction tips. That's right, I was once a malacologist and once even did laboratory work. Here is a CTAB protocol based on Doyle & Doyle (1987; Phytochemical Bulletin 19: 11-15) and a variety of other papers. I include some of our modifications (we developed our own protocol independently but merged it with a protocol from Dr. Robert Vrijenhoek's laboratory) and modifications from Dr. M.G. Harasewych (NMNH). We used this method on invertebrates, vertebrates, and bacculovirus pips. It works on invertebrates frozen or preserved in formalin/alcohol or DNE solution. It works on vertebrates frozen, preserved in alcohol, or dried skin samples. It has worked well for very small gastropods (half shell at that) followed by PCR, but we suggest you examine alternate techniques for extractions from blood or single hairs. There is a superior protocol for extraction from formalin preserved invertebrates, see my next post.


PROTOCOL

1. Grind 1-2 cubic millimetres of tissue in a 1.5 ml eppendorf tube with 300 ul of 2X CTAB buffer @ 60C (we often add a pinch of white quartz sand, -50 to 70 mesh, from Sigma or some ground glass to help grind snail foot tissue, tough fish parts, or mammal skin samples)
2. Incubate in water bath @ 60C for 30 min.
3. Add 300 ul of chloroform-isoamyl alcohol (96:4) and shake for 2 min.
4. Centrifuge @ 15,000g for 10 min.
5. Take aqueous and repeat chloroform-isoamyl alcohol extraction.
6. Label fresh eppendorfs and add 600 ul cold 70% EtOH, 25 ul 3M NaOAc, and the aqueous from the chloroform extraction. Mix by inversion.
7. Centrifuge @ 15,000g for 10 min.
8. Pour off EtOH and rinse pellet in 70% EtOH.
9. Centrifuge @ 15,000g for 5 min.
10. Pour off EtOH. Dry pellet in oven (60-70C) for 2-5 min. (not too long or pellet will dry out) (we have used a speedvac instead of an oven with no problems)
11. Re-dissolve the pellet in 25 ul of 0.1X TE and place in oven (60-70C) for 30 min. or overnight to get all DNA into solution. (resuspend in more 0.1X TE if a large pellet but remember that is always easier to dilute later than concentrate)
12. Verify quality and quantity using agarose gels, spectrophotometers, or fluorometers. (CTAB buffer does cause some shearing of DNA but we PCR from the extract all the time. The CTAB extraction also yields large quantities of good quality RNA. We have not found that the RNA interferes with PCR as reported in the literature. We have not tried blots on CTAB extracted DNA or RNA.)

2X CTAB Isolation Buffer

• 100 mM Tris-HCl, pH 8.0
• 1.4 M NaCl
• 20 mM EDTA
• 2% Hexadecyltrimethylammonium bromide (CTAB)
• 2% polyvinylpyrolidone (40,000 MW)
• 0.2% 2-mercaptoethanol -- add only immediately prior to use!

(We actually make the buffer from solids and do not adjust pH. For a 100 ml batch this takes 1.21 gm Tris, 8.182 gm NaCl, 0.74 gm EDTA, 2.0 gm CTAB, 2 ml polyvinylpyrolidone, 0.2 ml 2-mercaptoethanol, and water to volume. Take proper precautions when handling 2-mercaptoethanol. We have not examined the shelf-life of the CTAB buffer but have used a single batch for 2-3 months without problems.)