Ferland Louis H.
ferlandl at ERE.UMontreal.CA
Thu Feb 1 03:27:29 EST 1996
We make our own 1 cc columns of Sephadex G-50 in insulin needles,
equilibrate with TE, apply the sample (PCR, random-primed, riboprobe,
what-have-you) and elute 10-15 fractions of 100 ul with TE (we spin, but
care must be taken to not dry the resin). unless they are very short, the
probes come out in fractions 3-5 and the free nucleotides in 8-10. Those
probes hybridize quite well to Northerns/Southerns in our hands.
Give it a shot. It's easy and inexpensive. If money is not a concern, you
can buy ready-to-use columns (e.g. Pharmacia's NAP-10 columns, Sephadex
G-10, I believe).
On 29 Jan 1996, dbjerm at foothill.net wrote:
> Date: 29 Jan 1996 05:38:18 GMT
> From: dbjerm at foothill.net <dbjerm at foothill.net>
> To: methods at net.bio.net
> Subject: amplified inserts
> I am interested in how to get PCR-amplified cDNAs to hybridize cleanly
> to a Southern blot. I get clean insert bands on a 1% agarose gel, but
> the autorads have a low signal/noise ratio. I have tried several
> methods to remove dNTPs, buffers, etc from the propduct before labeling
> these inserts, but nothing has given satisfactory results. Anybody
> have a method that works well???
Dr. Louis H. Ferland
Centre de Recherche, Hotel-Dieu de Montreal
Dept de Nutrition, Universite de Montreal
Phone: (514) 843-2757 FAX: (514) 843-2719
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