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Sequencing PCR Products - Please Help
Posted by: schoening (IP Hidden, New member, 0)
Date: July 6, 2002 03:41AM
I have been purifying PCR products with a Qiagen kit in anticipation of sequencing them. However, when I run the DNA on an agarose gel to estimate the concentration, I still see primer-dimers. I understand that primer-dimer contamination will ruin the sequencing reaction results.
Surely I am not the first person to have this problem. I am working on using PCR conditions which use less primer and more template DNA. I would rather not gel-purify each PCR product because I need to sequence so many of them. Also, I think I will try a hot start for my PCR because I've read that it decreases primer-dimer concentration. Does anyone have suggestions? Has anyone tried sequencing a PCR product with some small primer-dimer contamination visible on the gel? Any help would be much appreciated.
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