isoamyl alcohol

Gys de Jongh GysdeJongh at planet.nl
Mon Mar 26 16:49:37 EST 2001


"David F. Spencer" <dspencer at is.dal.ca> wrote in message
news:dspencer-F875B7.17361622032001 at News.Dal.CA...
> In article <3.0.6.32.20010318081653.007bcb30 at cnmailsvr.nmsu.edu>,
> hroychow at nmsu.edu ("Dr. Hiranya S. Roychowdhury") wrote:
>
> > At 02:54 PM 3/18/01 +0100, Gys de Jongh wrote:
> >
> > >
> > >Hi,
> > >if you use sodium acetate the SDS will precipitate.
> > >The SDS stays in solution at high NaCl concentrations.
> > >See maniatis appendix
> > >--
> > >Gys

Hi,
got the flu , been away for a few days.
Here is the reference :

1) Molecular cloning
    Sambrook,J
    second edition 1989
    Volume 3
    Appendix E : Commonly Used Techniques in Molecular Cloning
    subject : concentrating nucleic acids
    subtitle : precipitation with ethanol or isopropanol
    page : E.10

quote : Sodium chloride (0.2M) should be used if the DNA sample contains SDS.
The detergent then remains soluble in 70% ethanol.  Sodium acetate (0.3M;pH 5.2)
is used for most routine precipitations of DNA and RNA. unquote.

[ See the problem in the original post ]
[ Subject: isoamyl alcohol ]
[ From: Ron Kim (rkim02 at emerald.tufts.edu) ]
[ Date: Wed 07 Mar 2001 - 16:31:44 GMT ]


Some time ago a had a solution of 10% SDS containing about 50 nanograms per
microliter DNA . This was standing on my lab table in a clear tube. I wanted the
DNA. I started by adding NaAc/HAc (3 M pH 5.2 ref 1 , page B.13). The tube
filled itself immediately with a large fluffy white cloud. One of those days I
thought , started reading and found ref 1. I repeated the experiment . This time
by adding NaCl 5M , final concentration 0.2M (ref1 table E.1 page E.11) In about
half of the tubes I saw a faint precipitation which went back in solution apon
gentle agitation. Support for the observation in ref 1 I think (?) I then added
2 vols of ethanol , cooled etc and finally got the expected small pellet which
proved to be rather clean DNA in subsequent experiments.

[Thought of Hiranya ]
[ Subject: Re: isoamyl alcohol ]
[ From: Dr. Hiranya S. Roychowdhury (hroychow at nmsu.edu) ]
[ Date: Sat 17 Mar 2001 - 13:19:09 GMT ]
[ With acetate you'll get unsoluble sodium acetate. ]
[ Hmmmmmmm! Interesting observation ... ]

> >
> >
> > Eh?
> > Dr. Hiranya S. Roychowdhury
> That sums up my reaction as well!
>
> I did a brief look through Sambrook et al. but couldn't find such a
> statement. Someone must be confused.
>
> Dave

This effect amazes me too . I think there may be confusing about what is
precipitating. I think we may safely conclude that this must be SDS and not
sodium acetate. There is no further ref or explanation in ref 1.
Annyone ??

[ Suggestion of Cornelius ]
[ Subject: Re: isoamyl alcohol ]
[ From: Cornelius Krasel (krasel at wpxx02.toxi.uni-wuerzburg.de) ]
[ Date: Thu 22 Mar 2001 - 08:49:25 GMT ]

I tried his suggestion today.
I added KCl 4M , final concentration 0.2M , to a 10% solution of SDS. This
resulted in a very solid precipitation. Repeated it with 5% and 1% SDS with the
same result. Seams Cornelius found a nice alternative solution of the problem.

--
Gys
If all else fails , read the directions :)






More information about the Methods mailing list