Sequencing problem
Martin Weinberger
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
weinb...@sc3101.med.buffalo.edu
Buffalo, NY
David M. Berman
>Dear Netters,
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
berm...@swmed.edu
Dallas, TX
Ron Kagan
weinb...@sc3101.med.buffalo.edu writes:
>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.
I would reccommend thermocycle sequencing with sequencing grade taq
polymerase, using any of a number of kits, such as USB's Delta Taq or
Taquence, or Promega's fmole kit. This method allows you to carry out
the annealing and extension steps at 70C, and you can also use deaza-dGTP.
Ron Kagan
rka...@ewald.mbi.ucla.edu