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Contamination in qRT-PCR RT-minus - band similar to cDNA, not sure what it is?


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#1 seven_doctor

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Posted 13 December 2014 - 07:56 AM

Hi everyone,

Hoped someone may be able to offer some suggestions.

In my qRT-PCR with SYBR, I keep getting a product in my RT-minus reactions for one primer set (a melt curve peak, though TM variable and different from positive, as well as band when i run reaction on gel, although band seems slightly larger than cDNA positive). The primers span introns, my RNA is DNase-treated and I get no products in reactions with other primer sets, suggesting the RT-minus samples are not contaminated with cDNA, nor do i get amplification in my NTC, suggesting no general contamination of primers/water etc. Im a bit stumped as to what is going on, and hoped someone may have a suggestion? Primer-blast shows my primers are specific to my gene, and no hits in genomic DNA database. Im thinking of getting the band in question sequenced to see what it is? 

Ive attached some files - The Log fluorescence qPCR amplification (positive in Brown, rest RT-minus), melt curve (positive in Brown) and the gel, which I hope will provide enough info!

Anyway, thanks for reading, and hope you may have some tips or tricks!

Thank you!

Attached Thumbnails

  • Log Fluorescence.PNG
  • Melt Curve.PNG
  • Gel.jpg


#2 nightingale

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Posted 14 December 2014 - 05:11 AM

hello seven_doctor, 

 

have you tried repeating the DNase treatment ? 


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