A good Oligolabelling protocol*****

Colin Rasmussen crasmussen at anat.med.ualberta.ca
Sun Jun 4 00:10:26 EST 1995


In article <3qq20b$1d7 at bubba.NMSU.Edu>, smori at nmsu.edu (Shahram Mori) wrote:

> Dear Molbionetters,
> I have been doing a few oligo-labelling reactions and they have been less 
> than acceptable. I would really appreciate it if somebody could post a 
> good oligolabelling reacion on the net. Thanks a million.
> (this oligo-labelling procedure is used for Northern Blots).
> 

We use the procedure outlined in the newer editions of the Maniatis
cloning manual. The procedure is based on the original work of Feinberg &
Vogelstein and works very well in our hands. We get our random hexamers
from Pharmacia, use Promega Klenow, and Amersham 32P-dCTP.  We typically
use about 50ng DNA (purified on LMP agarose) in a 25ul reaction incubated
at 37°C for 1-2 hrs.  The probe is separated from the unincorporated dNTPs
on G-50 minicolumns and we usually get 60-80% incorporation per 50uCi 32P
added.

In regards to helping you with your problem, what do you mean by less than
acceptable. Many factors can affect the efficiency of labelling and it
would be useful for you to be more specific.

Colin



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