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PCR on PCR of cDNA smear - (Dec/12/2005 )

I don't really know whats going on but maybe somebody has some hints. I am using cDNA to get PCR product I want to use as standard in real-time PCR. I do my PCR and I get (a weak) product. To increase the DNA I made an additional PCR of the former product, which I gel purified. Instead of getting a stronger band of the original product it always produces smear (up to very high molecule sizes up tp 20 kb from original 390bp in 30 cycles). I checked the same gene (cDNA) with a different primer set and get the same. I am going to sequence some of the different size products to check out if I just had somehow overlapping sequences which lead to a chain of the original product.
But as I used an elongation time of 35 sec in the PCR reaction I actually can't see how I get high molecular products......
If anybody has suggestions (contaminations? stupid mistakes I might have done...) I would be happy. It only happens with one particular gene. I am not sure if maybe some splicing events could explain it....


I would check for overlap.

You underestimate the processivity of Taq polymerase. 30 seconds is way enough to extend ~1000 bp.