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PCR smear for genomic samples

PCR smear DNA

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4 replies to this topic

#1 sofialia

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Posted 04 June 2012 - 12:29 PM

I am running PCR for genomic samples with DNA that was extracted from embryos. This is a routine PCR that has worked great in the past, meaning I haven't changed primers or reaction conditions. In the latest PCR that I have completed, my the PCR product for the samples smeared on the agarose gel (1% TAE). However, the control samples that I tested (DNA that I know works) didn't smear on the gel and had a nice solid PCR band at the expected size. I'm thinking that the new DNA is either more concentrated, contains more impurities, or has degraded.

Another side note, I have also used these same samples using a different set of primers and the PCR bands came out very clean and bright. Any suggestions or ideas are highly welcomed!

Thanks for any help.

#2 Curlis

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Posted 04 June 2012 - 12:43 PM

I am running PCR for genomic samples with DNA that was extracted from embryos. This is a routine PCR that has worked great in the past, meaning I haven't changed primers or reaction conditions. In the latest PCR that I have completed, my the PCR product for the samples smeared on the agarose gel (1% TAE). However, the control samples that I tested (DNA that I know works) didn't smear on the gel and had a nice solid PCR band at the expected size. I'm thinking that the new DNA is either more concentrated, contains more impurities, or has degraded.

Another side note, I have also used these same samples using a different set of primers and the PCR bands came out very clean and bright. Any suggestions or ideas are highly welcomed!

Thanks for any help.

Mostly smear in PCR is because of too much template has been take for PCR. Dilute your template by TE buffer. Do dilutions of 1/10, 1/50 ,1/100, 1/200, 1/300, 1/400, 1/500. I think after dilution it will work.

#3 AquaPlasmid

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Posted 04 June 2012 - 10:58 PM

Did you make your own master mix? High [Mg] could give PCR smear. But your positive control and other primer pair worked fine. That rules out the Mg possibility. Where was the smear located, near the well (amplification of Taq contaminated bacterial gDNA) or below your target (template contaminated with PCR inhibitors)?

#4 sofialia

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Posted 05 June 2012 - 02:06 PM

The smear was located near the well. I ran another PCR today with these primers and had the same result, although this tim the smear was located farther down with a gap in between the well and the smear. This problem only happens when I switch primer pairs, and the same DNA works perfectly fine with the other primer pairs that I have.

#5 AquaPlasmid

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Posted 06 June 2012 - 05:04 PM

Your new primers likely annealed to and amplified the bacterial DNA. You may try increase the annealing temperature, or re-design and order another pair of primers. We found most polymerases have bacterial gDNA contamination, when we tested them with isothermal amplifications.





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