Primer extension method of ALF express

Steven Goldberg goldberg at bms.com
Wed Oct 23 15:03:24 EST 1996


In article <3266AE72.32EA at molbiol.uct.ac.za>, Ant Smith
<ant at molbiol.uct.ac.za> wrote:

> Hi
> 
> Can anyone provide me with a protocol for doing primer extensions using 
> the ALF express automated DNA sequencer. I have not seen any kits for 
> this provided by Pharmacia. 
> 
> I have the unlabeled primers so a method using incorparation is what I 
> require.
> 
> Thanks
> Ant Smith
> Department of Microbiology
> University of Cape Town
> South Africa

Ant:  I have used primer extension with the ALFexpress.  Cy5-dATP is
incorporated during the synthesis.  There are specific rules about where
the Cy5-dATP is incorporated: 1)  The label should be incorporated 1-5
bases after the primer 2) No more than one Cy5-dATP molecule should be
incorporated within the first 10 bases after the primer.  Therefore, you
can't just use any primer; it has to be planned carefully.  However, it
does work quite well and I routinely get 500-650 bases of clear sequence
data per reaction with dsDNA.

Your best bet is to contact Roberto Rodriguez at Pharmacia (USA) at
1-800-526-3593 or at robertoalf at aol.com for more information and
protocols.

Hope this helps.

Steve



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