Seems to me that you are adding far to much template to your sequencing
reaction which leads to excessive signal strength when sequencing resulting
in huge peaks with flattened tops. The appaerance of other peaks under them
is also due to too intense signals where the matrix correction is not
capable of completely correcting this.
Indeed this leads to a lot of N calls.
So it seems that you will have to add far less PCR product to your
sequencing reactions.
Hope this helps.
Greetings
Dieter
>
>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
>
>
>
>
>
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* Dieter Deforce *
* Lab Pharmaceutical Biotechnology *
* Harelbekestraat 72 *
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* University of Gent *
* 9000 Ghent *
* E-mail : Dieter.Deforce@rug.ac.be *
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