You may want to check the signal numbers for your sequencing reaction. My
guess is that they are very high - above 1000?. It is very easy to have
too much DNA when sequencing from PCR reactions. I think what you are
seeing is overloading of the detector with fluorescent signal, and
"pull-up" of the other colors in the spectrum. My suggestion is to cut the
amount of DNA in your reactions. If you have clean template, PCR reactions
can give as accurate sequence as plasmid templates.
At 10:20 PM -0400 7/23/98, robert obrien wrote:
>I have recently begun sequencing 200bp pcr products on the 377XL and have
>been noticing the following abnormalities: Peaks have a flat top appearance
>to them and there are peaks under larger ones. Is it customary to get a lot
>of N calls? What% is acceptable to get?
>
>
>Any Suggestions will be greatly appreciated
>
>
>Thank You
>
>Robert O'Brien
Mary Kay Dolejsi, Ph.D.
Manager, Biotech lab
marykay@fred.fhcrc.org
phone: 206-667-4470
fax: 206-667-6497
Mail address:
Fred Hutchinson Cancer Research Center
1100 Fairview Ave N. A1-162
PO Box 19024
Seattle, WA 98109-1024