From owner-biotechniques@net.bio.net Mon Nov 13 22:00:00 1995 Path: biosci!biosci!not-for-mail From: Hinrich Schulenburg Newsgroups: bionet.journals.letters.biotechniques Subject: CTAB-DNA-isolation-procedure Date: 14 Nov 1995 07:23:35 -0800 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 71 Sender: daemon@net.bio.net Approved: MMCCARTHY@BIOTECHNET.COM Message-ID: <488bjt$ato@mserv1.dl.ac.uk> hi everyone, 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 CTAB-DNA-isolation procedures. 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 about that? 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, Hinrich .