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digested PCR product - smeary band?

digested PCR product - smeary band? - Agarose Gel Electrophoresis Forum

digested PCR product - smeary band? - Agarose Gel Electrophoresis Forum. A forum to discuss electrophoresis staining, troubleshooting, and development of films for DNA and RNA gels.


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Old 02-15-2011, 10:46 AM
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Default digested PCR product - smeary band?



Hi all!

I have a question - is it possible that my digested PCR product appear as not clear band due to protein content in reaction?
I digested plasmid, and it appeared as quite nice bands on gel. Then I use this digested fragment of plasmid in fusion PCR with 2 fragment of genomic DNA (both of them were too extracted from agarose gel by commercial kit) and PCR product again digested. After this second digestion I run gel electrophoresis and I had smeary band in a fusion PCR product size and smear around the area where gDNA fragment should be found. May the protein from "digestion mix" (or protein/DNA ratio) affect product or template?

In original article authors recommended phenol:chloroform extraction and ethanol precipitation before every gel extraction, but we avoided this in attempt to minimalize losses.

Thanks lot!

Last edited by majihec; 02-15-2011 at 10:48 AM.
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