Sequencing PCR Products - Please Help

laura_schoening at hotmail.com laura_schoening at hotmail.com
Sat Jul 6 02:41:12 EST 2002


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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