Sequencing PCR products
Allison Pinder (pinder@mail1.ciwemb.edu)
24 Jul 1998 10:36:47 -0400
Robert- I agree that you must be using too much template as a cause of the
flattop peaks. With regard to the peaks over peaks, does it look like two or
more sequences being read in the same lane? Potential causes are mixed PCR
products or not getting rid of the old PCR primers before adding new
sequencing primers. We gel purify pcr products before sequencing to avoid
both of these problems, this can also give you an eyeball test for the
quantity of product you are dealing with. Occasionally PCR products are mixed
even when they appear on a gel as a single band (especially if someone in a
hurry did not run the gel out very far...). It should be possible to get
excellent sequencing from PCR products, probably better than with the huge
constructs people want to use these days. Good luck.
Allison Pinder
DNA Sequencing Core Facility
Department of Embryology
Carnegie Institution of Washington
115 W. University Parkway
Baltimore, MD 21210
410 554-1207
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