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Help! Smear in Real-Time PCR Product


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#1 silverphoenix

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Posted 15 June 2011 - 01:40 PM

Hi,

I am trying to perform qPCR on cDNA samples from mice. I performed my cDNA synthesis with Superscript Vilo (Invitrogen), which is based on random priming. I have some samples in which I observe non-sigmoidal amplification plots, these samples have very low Ct counts (See attachment). And if I run these samples on an 1% agarose gel I observe smearing in addition to the PCR product of expected size (See attachment). For my qPCR I used 1:10 diluted cDNA with Quantitect SYBR Green Kit (Qiagen).

What are these smears on the gel, and how can I get rid of them?
Any ideas or suggestions or solutions will be greatly appreciated

THANK YOU!!

Attached Thumbnails

  • L32_cDNA_1hr_0615112.jpg
  • 10fold dilution - GF.JPG
  • Melting Curve - All Samples.JPG


#2 pcrman

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Posted 08 July 2011 - 08:35 PM

It appears to me you either have genomic DNA contamination or RNA degradation. Can you run your RNA on an agarose gel to check both?




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