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Two bands in high-annealing-temperature samples

PCR gradient annealing temp. doubleband

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#1 torte

torte

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Posted 04 November 2011 - 05:13 AM

Hi,
I ordered new primers and performed PCR with gradient of annealing temperatures (8 temperatures). Surprisingly, I got two bands in reactions with the highest ann. temperature. The lower band weakend and disapeared with the decrease of annealing temperature. There may be some alternative splicing according to our previous results. The only explanation I can think is that in reactions with lower annealing temperature, the amplification of the more abundant upper band overcharges the amplification of the lower band. What do you think? I appreciate any suggestion Posted Image
T.





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