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