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 

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
Biochemical Instrumentation
Zimmermann at EMBL-Heidelberg.DE

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