CTAB protocol for mollusc DNA
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
- Put approximately 200 mg (wet weight) of gill tissue in a 1.5 ml
- Add 600 µl of CTAB buffer.
- (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.
- Incubate for two hours at 65° C.
- Freeze at -20° C for 30 minutes.
- Thaw at 65° C for 15 minutes.
- Suck the tissue through a wide-bore 1 ml pipette tip repeatedly, to
disrupt the tissue.
- Incubate for two more hours at 65° C.
- 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
- Precipitate with 300 µl cold isopropanol. Add the isopropanol,
mix by gently inverting the tube a few times, then put on ice for 15
- Spin in a microcentrifuge for 30 seconds (don't spin for longer than
this, as the pellet will be hard to resuspend).
- Remove and discard the liquid without disturbing the pellet.
- 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
- 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.
- 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
- 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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This page was last revised July 21, 2002. Its URL is