Sequencing PCR Products - Please Help
b.collet at abdn.ac.uk
Mon Jul 8 05:07:33 EST 2002
I recently sequenced PCR products with success after cutting the band out of the gel and purification using the Qiagen Gel extraction Kit with a final elution in water instead of Elution Buffer. This would solve you contamination problem with primer dimers.
laura_schoening at hotmail.com wrote:
> 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.
Dr Bertrand Collet
Fish immunology group
Aberdeen AB24 2TZ
Scotland - UK
Tel (office): (+44)(0)(1224)27 3796
Tel (lab): (+44)(0)(1224)27 2870
Fax: (+44)(0)(1224)27 2396
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