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QRT-PCR intermittent melt curve problem

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#1 cashlern



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Posted 10 August 2011 - 11:57 AM

I have been having an intermittent issue with my cyber green QRT assays. The same set-ups previously have shown perfect melt curves. My last PCR displayed good amplification plots yet the dissociation curves for both my target gene and actin didn't work. The same primer sets previously had given perfect melt curves but my latest reaction shows melt curves with multiple positive and negative peaks for both the cyber and rox dye. It just looks like "noise." I ran all my samples on agarose which showed single bands of expected size. How could I get clean amplified products at about 90-100 bp's yet a dissociation curve that fails for each primer and replicate? Remember these same primes gave me otherwise perfect melt curves last week. I use a stratagene mx3000p machine and Thermos 1-step QRT PCR kit.

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