Fluoro-dATP sequencing kit problem

Zimmermann at EMBL-Heidelberg.DE Zimmermann at EMBL-Heidelberg.DE
Thu Apr 6 18:40:12 EST 1995


> Now there is a kit utilizing fluorescein labelled dATP:s, but it doesn't seem
> to work at all. First comes the strong signal caused by unincorporated label
> and then nothing. The signal does prove that there is fluorescein present.
> Only a few attempts to make it work have been made this far, but I'd like to
> know if anybody has been able to make it work? Maybe it is just a bad lot or
> something like that? 
> 
We are using fluorescein-dATP in a home made kit, since roughly a year on a 
routine basis, here at EMBL. Actually we are using labelled primers only in a 
few rare cases.

I don't know if You sequenced ds or ss DNA. The occurance of huge stop in 
reaction indicates normally a neutralisation problem in double stranded 
sequencing.

Efficient labeling in a sequencing reaction is dependent on the overall primer 
ability and the position of the next A after the primer. This first 'A' should 
be at least within four bases after the primer.

If Your are interested, we can communicate adeteiled protocoll.

===============================================================================
Juergen Zimmermann
EMBL
Biochemical Instrumentation
Zimmermann at EMBL-Heidelberg.DE
===============================================================================



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