cwen at aux.btny.purdue.edu
Tue Nov 14 12:22:03 EST 1995
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.
CTAB is the best method I have ever used to isolate DNA/RNA.
>i) Is it possible to autoclave a CTAB-solution or does one 'destroy'
CTAB by doing so ?
Usually, I prepare CTAB solution(s) using DEPC-treated water without
autoclaving. However, I did not have problem to use autoclaved CTAB
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.....
It's a correct procedure. The original procedure used 0.7M NaCl/1%
CTAB..1XTris, EDTA...., Homogenize dehydrated tissue with 1XCTAB
extraction buffer, then a chloroform extraction is followed. After
Chloroform, the sup is precipitated by addition of 1Xvol of CTAB ppt
buffer which is 1% CTAB/Tris/EDTA without salt. Under the condition of
low salt, <0.4M NaCl, the CTAB/nucleic acids complex may precipitate and
leave solubilized proteins and polysaccharides in the solution.
Therefore, in your procedure, 2XCTAB is used for cells, assuming that
the cells contain 100% water. In my knolodge or experience, the actual
CTAB conc is not critical during this procedure, as long as it's above
>iii) What is CTAB doing in detail? I read a couple of more profound
papers about CTAB-DNA-isolation-procedures
CTAB is a detergent, as well as a compound similar to an
anion-binding reagent. Any negatively charged molecular may be bound
with CTAB. I do believe that CTAB also precipitate some negatively
charged proteins and polysaccharides(based on my personal experience).
Under low-salt condition, those CTAB-bound molecular may precipitate.
It's why you may observe a bigger pellet after this ppt procedure, while
a small ppt is observed after EtOH ppt.
>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)
I do not use DTAB procedure, and do not know what DTAB is. However,
if it is not a detergent, the DNA may be degraded.(I do not know the
chemical property of DTAB, just a guess).
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.
I have tried a lot of procedures to isolate RNA and all of them did
not work. I would say that the polysaccharides content is hight in the
tissue I am working with and DNA/RNA which are negatively charged might
bind to positively charged polysaccharides and trapped in the big
insoluble polysaccharide pellet. CTAB may work as an anion exchanger,
therefore exchange the DNA/RNA from the polysaccharides.
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