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PCR smear

PCR smear - PCR - Polymerase Chain Reaction Forum

PCR smear - PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory.


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Old 08-03-2011, 06:14 PM
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Default PCR smear



I was doing PCR for a month day everything was going ok suddenly I saw smearing in the second time I was doing PCR using my primary PCR product. I started series of troubleshooting later even my primary PCR is showing smearing. The smear appears right from the wells. So far I have changed the PCR mastermix, new primers were diluted and used, new water, the only thing I didn't try is to use new running buffer. Now using positive control during troubleshooting I am seeing smearing around the right band size tough I don't see the band clearly.

What do you suggest? Please need help urgentlyyyy. Thank UUUUU
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Old 08-20-2011, 05:21 AM
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Default Re: PCR smear

Smearing can be caused by high concentration template used for PCR or degradation of product. For 2nd PCR using 1st PCR amplicon as template, its common to see smearing on the gel after PCR due to too much template used. You can lower the amount of template used for PCR. Recycled running buffer will have different concentration as initial and salt deposition will cause conductivity difference, hence, should change the buffer time to time too. good luck
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Old 08-20-2011, 12:53 PM
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Default Re: PCR smear

Better upload the Gel picture so th6at one can h6ave better idea wh6at you are discussing5 about.

And don't use PCR product as PCR template. Most of the time i wouldn't work because it not stable like normal DNA ( if you are using it as standard, Never use it).

I think Smear was just degraded PCR product which you used as template
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