Cycle sequencing anyone?
jkropp at itsa.ucsf.edu
Thu Mar 27 20:53:52 EST 1997
In article <3334A8B1.15BC at utoronto.ca>, Ed Marsden
<ed.marsden at utoronto.ca> wrote:
> For the sake of simplicity, I've attempted to cycle sequence both the
> commercial plasmids and my own ds isolate as opposed to using the
> standard protocol (we're using the Boehringer Taq polymerase kit and DIG
> detection). (snip)
I fooled around with DIG detection a few yr ago, before the "beta testing"
results had been incorporated into the new protocols. Results: lotsa black
film. So I bagged it.
Couple of yr ago I decided to try non-R/A sequencing again, using a
biotinylated primer and detecting with the NEBL Phototope system. Worked
fine. Last yr I ported those same primers to cycle sequencing protocols--
Thermosequenase from Amersham and Sequitherm from 5'>3'--and the results
were just as acceptable. I sequence PCR products of human genomic DNA BTW.
It is very sensitive, and you can get away with a less-than-pristine template.
If you decide to try it let me know. I have a few tricks to ease the pain...
"If it ain't broke, it needs more features"-plagerized
More information about the Methods