Mollusc DNA Extraction - Formalin Perserved Samples

When extracting from fresh or frozen invertebrate specimens, we usually use the CTAB protocol (see my previous post). In the past, we also used this protocol for working with specimens that had been stored in formalin for some period before being transfered to ethanol but the results were hit and miss. The Etter/Rex group at UMass Boston developed a much superior protocol (Chase et al. 1998). It works incredibly well for formalin preserved invertebrates and we highly recommend it. The resulting DNA is very workable for PCR, although formalin still places limits on product lengths. It is basically the France and Kocher (1996) protocol but with the use of a silica-based column instead of ethanol precipitations. Here is our version of the protocol. Please cite Chase et al. (1998) when using it.

The protocol was centered on the QIAamp Tissue Kit. I don't know if this still exists in the same form, but take a look at the QIAamp DNA FFPE Tissue Kit. Be sure to order extra Buffer ATL - it can be ordered separately. Also be sure to prepare the solutions as described in the manual.

1. Mince 250 mg tissue, add 200 ul Buffer ATL, place at 55C for 24 hours.
2. Add 5 ul of 50 mg/ml Proteinase K and an additional 95 ul Buffer ATL. Place at 55C for 72 hours, in a mild shaker or with occasional vortexing.
3. Add 200 ul Buffer AL, mix by vortexing and incubate at 70C for 10 minutes.
4. Add 210 ul of 95% Ethanol, mix by vortexing.
5. Add mixture to column, centrifuge at 8000 rpm for 1 minute.
6. Place spin column in fresh tube, discard used collection tube.
7. Add 500 ul Buffer AW to column, centrifuge at 8000 rpm for 1 minute. Place spin column in fresh tube, discard used collection tube.
8. Add 500 ul Buffer AW to column, centrifuge at full speed for 3 minutes.
9. Place spin column in fresh tube, discard used collection tube.

Read the manual carefully to decide which elution strategy is best for your sample. Here are some options:

• Good Tissue or Lots of Tissue: Elute twice with 200 ul Buffer AE preheated to 70C. Incubate at room temperature for 1 minute before spinning for 1 minute at 8000 rpm. This is the manual's suggested approach. If you think DNA is rare, use less Buffer AE.
• Minute Specimens or Heavily Formalized Tissue: Add 50 ul of 70C preheated Buffer AE. Incubate at 70C for 5 minutes. Spin for 1 minute at 8000 rpm. Do not perform a second elution.