Sequencing problem

[David M. Berman]

weinberger at sc3101.med.buffalo.edu (Martin Weinberger) writes:

>Dear Netters,

>Using Sequenase 2.0, I am having difficulty resolving some G residues distal
>from the primer.  I have tried using a higher temperature during the extension
>and termination reactions with the USB-recommended glycerol-tolerant buffer and
>substituting dGTP with dITP.   These steps have not  helped.  Can someone
>suggest further modifications to the Sequenase protocol or recommend an
>alternative sequencing method that will allow me to cleanly resolve the
>nucleotide sequence.
>Thanks in advance for any and all advice.

>Martin Weinberger
>weinberger at sc3101.med.buffalo.edu
>Buffalo, NY


I've had good luck with dITP substitution but running a hot formamide
gel also helps with compressions.  Use 30 - 40% formamide in an
otherwise normal TBE/urea gel.  Prerun the gel at about 70 Watts for
15-20 min and load. You can't dry these gels down directly so either
use 32P-dATP in your sequencing reaction or fix and soak your gel b/4
drying down (I've always done the former).  Good luck.

David Berman
berman02 at swmed.edu
Dallas, TX