amplified inserts

[Ferland Louis H.]

Dear dbjerm,

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).

Louis F.


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