washing alcohol-preserved sample before DNA isolation... H2O, TE, or? - (Dec/10/2008 )
I've found very little regarding alcohol-preserved samples (EtOH) and if and how they should be washed before nucleic-acid isolation. I've found so far that one SHOULD wash it in TE buffer (no concentration specified!), that one can wash it in sterile H2O.
I have several questions:
1) what is best to wash the alcohol? or does it have to be washed at all?
2) does it depend on the protocol? if so, what should I do for phenol-chloroform extraction?
3) if TE, what is the reccomended concentration?
4) if H2O, doesn't it damage the tissue, and potentially the DNA? is it necessary to make a serial dilution from alcohol to H2O?
5) can I extract RNA from alcohol-preserved samples?, and if so:
6) what's the best procedure?
TE is (almost always) at the defined concentrations of 10 mM Tris-HCl, 1 mM EDTA, pH 7.6, so people don't specify the concentration. They may say "10x TE" or "100x TE."
Washing with TE would be my choice, since it tends to preserve isolated DNA, but it probably makes little difference if you use water or PBS. You should not use alcohol, which will mix with the phenol in a subsequent phenol-chloroform extraction. Small amounts of alcohol in the cells are probably not a problem, assuming you have an excess of phenol in the extraction. No serial dilution should be necessary. Yes, you can extract RNA, assuming it is still there. Trizol, or one of the kits would be my first choice.
Well, that's exactly what I mean. I've been using 100X TE, but actually never read / heard wether I should use another dilution. I guess as it is such a good buffer, it doesn't really matter, am I right? I was just wondering if it would interfere with the lysis...
Thanks a lot, I'll stick to the TE, then.
When people say "TE" they mean 1x TE. The 100x TE would not be my first choice to wash cells in, as its ionic concentration is very high. But it would probably work.