EtOH or Isopropanol for DNA precipitation
mbrow at twt.com
mbrow at twt.com
Mon Sep 21 17:11:15 EST 1998
In article <199809101941.NAA53802 at nestor.NMSU.Edu>,
hroychow at NMSU.EDU (Hiranya Roychowdhury) wrote:
> At 05:06 PM 9/9/98 -0400, Michael Santos wrote:
> >Hello everyone.
> >I thought this might be a good forum to ask a lingering question I have on
> >DNA precipitation. The protocol I follow for extracting Arabidopsis DNA
> >requires isopropanol to *selectively* precipitate high molecular weight DNA
> >(I have no idea for the basis of this claim; i.e. why wouldn't ethanol do
> >the same thing?). What is the reason for the establishment of isopropanol,
> >as opposed to ethanol, as the default alcohol for the initial precipitation
> >of DNA? (e.g. even protocols for plasmid preps specify isopropanol DNA
> >precipitation followed by ethanol washes) Thank you for taking time to
> Topic has been discussed more number of times over the past several
> years than I'd care to count. This is one of those items in mol. biol.
> protocol manuals that get lost amongst heaps of "modifications" and
> There are two reasons that justify 2-propanol use: 1. You only need
> to achieve 50% alcohol concentration as opposed to the 67-70% needed with
> etoh. 2. Consequently, less of the associated salts are co-precipitated.
> Hence, if the volume of the sample is not too much to handle in a single
> tube and if there is not much salt in the sample, any one of the two
> alcohols will do just fine.
> That 2-propanol is used to " *selectively* precipitate high
> molecular weight DNA" is a myth. It seems to me that the English language
> has become even more equivocal in its Scientific usages, and the author did
> not really mean to exclude ethanol from the distinction. That the sentence
> would read that way, is lost on most these days.
> Dr. Hiranya Sankar Roychowdhury
> Plant Genetic Engineering Lab.
> New Mexico State University
> Las Cruces, NM 88003
> Ph. (505) 646-5785
> hroychow at nmsu.edu
Selective precipitation is not mythic, at least not when you are working with
really small DNAs. Selective precipitation has been used for years to remove
primers and dNTPs from completed PCRs. It is particularly effective if the
solution is made 2M ammonium acetate, and the precipitation is performed at
room temp with 1 volume of isopropanol. This combination of salts and
alcohol is very inefficient at precipitating nucleic acids below about 100
bp, and the results are readily verified by gel analysis of aliquots before
and after precipitation. In contrast, a standard ethanol precipitation
(i.e., 2.5 vols of 100% added to either 0.3 M sodium acetate or 2M ammonium
acetate) yields a much higher return on the lower molecular weight materials.
The use of ammonium avoids precipitation of the dNTPs whether EtOH or
isopropanol is used, so the real difference is in the elimination of the
primers. The effect is particularly striking if the PCR is performed with
labeled primers because the PCR background is so much more visible than it is
when a gel is stained. So, this method works very well but is not recommended
for short amplicons.
The use of carrier, either high molecular weight DNA or glycogen, reduces the
selectivity, so you may well be correct that there is little benefit of
isopropanol use when preparing genomic DNA, which would certainly be a very
effective carrier to bring down smaller species.
My two cents....
Mary Ann Brow
Third Wave Technologies, Inc.
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