When OF(1ul) and outer OR(1ul) primers and IF(0.5 ul) and OR( 0.5 ul ) are used , it gave faint control band .And when only OF and OR primers are used it gave bright control band .How can it be optimized to get bright control and wild band ?
With allele specific pcr I would start with a normal and a homozygote mutant control sample and run temperature gradients on each sample with each primer set separately. There should be temperatures at which one sample amplifies and the other does not for each primer pair. these temperatures can be used to measure the presence of each allele in the presence of the other allele even if the reactions may need to be run in different tubes if these temperatures are too different due to the design of your primers
You can try adjusting the ratios of the different primers, make sure the extension time is long enough for the larger product, optimize the annealing temp. etc.
Or, you can be practical about this and you can set up the two reactions in different tubes. Sometimes you just can't get reliable duplex PCR.
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