in our lab, we are studying isopod phylogeography using DNA
sequences. However, DNA isolation from isopods turned out to be quite
difficult. We showed that these animals contain nucleases
of high activity, and next to that also Proteinase K-inhibitors.
To overcome all such problems, we have tried various DNA-isolation
methods, and we found a CTAB procedure to work best.
Nevertheless, we have the impression that particularly this procedure
could work better. This might be due to unexperience with
I would therefore be very grateful if someone of you could give me
some advice on the following:
i) Is it possible to autoclave a CTAB-solution or does one 'destroy'
CTAB by doing so ?
ii) We use a 2%CTAB-solution (including 1.4M NaCl, and also
EDTA, Tris-HCl, and beta-Mercaptoethanol) for lysis,
we then extract samples with 1x volume of Chloroform/Isoamylalcohol.
Thereafter, we specifically precipitate DNA with 3 volumes of a 1%
CTAB-solution (a pure 1%CTAB-solution without any other compounds).
(At this step, we should have a NaCl concentration of 0.35M and a
CTAB-concentration of about 1% !??), we then pellet the
CTAB-DNA-complex and resuspend it in high-salt-TE, finally followed
by a conventional DNA-precipitation with 100% ethanol.
However, I am not completely convinced that for the specific
DNA-precipitation, I am using the right CTAB or salt concentrations.
Does anyone of you know at which salt concentrations, CTAB
can be used to specifically precipitate DNA? What happens if salt
concentrations are slightly above the upper limit? What happens if
salt concentrations are far too low? Is CTAB-concentration at this
step of any importance (e.g. could I also use a 2% or a 0.5% pure
CTAB solution in order to precipitate DNA or does that cause
coprecipitation of contaminants)? Do you know of any detailed papers
iii) What is CTAB doing in detail? I read a couple of more profound
papers about CTAB-DNA-isolation-procedures (e.g. that one from Rogers
& Bendich or Shivij, Rogers & Bendich), but I did not find any
information why CTAB specifically binds to proteins at high salt
concentrations, and to nucleic acids at low salt concentrations. So
how does it do it? Do you know of any references about it?
iv) we also tried a DTAB-DNA-isolation procedure which didn't work
with isopods (we used a DTAB buffer for lysis, containing 6% DTAB,
EDTA, Tris-HCl, and NaCl, but no beta-Mercaptoethanol). So what is the
difference between the action of DTAB and CTAB?
v) in order to isolate DNA, we tried conventional procedures using a
lysis buffer containing EDTA,Tris-HCl, SDS, NaCl, Succrose and
Proteinase K (all at different concentrations), we tried a
Chelex-100-procedure, we tried boiling in different buffers, we tried
comercial DNA-isolation kits. But only the CTAB-procedure (using a
2%CTAB-buffer with 2% CTAB, EDTA, Tris-HCl and beta-Mercaptoethenol)
produced reproducible results. What does such a finding tell me about
isopod DNA, or about isopods in general? E.g. does this argue for a
strong bond between proteins and DNA which is only broken up using
CTAB (therefore, in all other approaches, DNA is 'extracted out', together
with the proteins to which it is bound) ?
I'd be very grateful if someone could give me an answer to
some of my questions. I would also be very interested if someone
is/was confronted with similar problems.
Many thanks in advance,