This protocol yields large quantities of DNA, suitable for the ARRF technique. It works well on gill and mantle tissue from mussels and oysters. Mollusc tissues contain large amounts of mucopolysaccharides, which interfere with many other DNA preparation protocols. I imagine this protocol would work well on other animals, whether or not they contain mucopolysaccharides. Plants, fungi and prokaryotes have their own difficulties, and since I don't have any experience with them, I can't offer any advice.

Some DNA preparation techniques use proteinase K to digest the tissue. I have found that adding proteinase K to this protocol is aesthetically pleasing, because the tissue completely dissolves in a few hours, but enough proteinase K is carried through the extractions that it prevents the ARRF technique from working well.

Some DNA preps suggest that you dry the final pellet for a few minutes, because that makes it easier to resuspend. I find that if I don't dry the pellet for a day or more, enough alcohol or chloroform remains that it interferes with the ARRF technique. Even after this long drying period, the pellet resuspends well if left in buffer overnight.

1. Put approximately 200 mg (wet weight) of gill tissue in a 1.5 ml microcentrifuge tube.
2. Add 600 µl of CTAB buffer.
3. (Optional).Mash the tissue by hand with a pestle. This is not necessary for bivalve gill tissue, which has a large surface area, but it would probably be helpful for many other tissue types. If you do this, add just 200 µl of the CTAB buffer, mash, then add the remaining 400 µl of CTAB buffer. Otherwise the tube will overflow when you put the pestle in.
4. Incubate for two hours at 65° C.
5. Freeze at -20° C for 30 minutes.
6. Thaw at 65° C for 15 minutes.
7. Suck the tissue through a wide-bore 1 ml pipette tip repeatedly, to disrupt the tissue.
8. Incubate for two more hours at 65° C.
9. Do three extractions with 500 µl chloroform/isoamyl alcohol. (The solution is 24 parts chloroform, 1 part isoamyl alcohol.) For each extraction, add 500 µl of chloroform/isoamyl alcohol, vortex for a few seconds, let sit for a couple minutes, then spin in a microcentrifuge for 3 minutes. Then carefully suck up most of the upper layer, while trying not to get the goopy stuff at the interface, and transfer it to a new tube.
10. Precipitate with 300 µl cold isopropanol. Add the isopropanol, mix by gently inverting the tube a few times, then put on ice for 15 minutes.
11. Spin in a microcentrifuge for 30 seconds (don't spin for longer than this, as the pellet will be hard to resuspend).
12. Remove and discard the liquid without disturbing the pellet.
13. Wash two times with 1 ml of cold 70% ethanol. For each wash, add the ethanol, gently invert the tube a few times, spin in a microcentrifuge for 15 seconds, then remove and discard the liquid without disturbing the pellet.
14. Air dry for one to several days (although the pellet may look dry after a few minutes, traces of alcohol or chloroform remain and will interfere with the restriction digests). I usually leave the tubes in a drawer, with the caps off, over a weekend.
15. Add 100 µl of TE, then let it sit in the refrigerator overnight or longer. Then suck through a pipette tip several times to resuspend the DNA.
16. Use 20 µl for each ARRF reaction.

When a small aliquot is run on a 1% minigel, the DNA should form a bright band near the top of the gel. Some shearing is acceptable, as long as most of the DNA is thousands of bp long. You can't test whether the DNA will cut with rare cutters without doing the whole ARRF procedure, so test it by cutting it with four cutters; if most of the ones you try cut it, that's a good sign.

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Send comments to John H. McDonald (mcdonald@udel.edu).

This page was last revised July 21, 2002. Its URL is http://udel.edu/~mcdonald/ctab.html