When I was doing PCR amplification of a cyt b gene of 1200bp in size and was getting positive bands of about 1000 bp on agarose gel at an annealing temperature of 53 degree celsius. But later when doing the repeated amplification of the gene using the same condition with a high quality DNA smeared bands are forming on gel without any distinct shape or size. I have attached a picture of the bands which I got on my 1.5 % agarose gel today. What could be the reason for this? Is it due to the degradation of primer or any other reason? please help.
Are there repeats within the DNA you are amplifying? If so, the primers may be annealing in more than one place, providing a large number of continuous bands.
I don't know what the Tm is of your primers but I would try a higher temp for annealing. It should be at or below the Tm of your primer.
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