In article <CI79M6.
...@acsu.buffalo.edu> Martin Weinberger,
weinber
...@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
