I have 2 different pairs of Primers, the annealing temperature of the 'reference pair' is 54C, while the temperature of the other 'test pair' is 60C.
I'm running my experiment on the device of Applied Biosystems '7500 Real-Time PCR System'. Thanks in advance.
Hi Sarah
I don't think the ABI 7500 has the gradient option. You should use separate reaction.
Hi, yes an idea with gradient is good, but if you have more than 12 samples for 1 run it could be difficult to work. Because of many runs. The best way are separate runs.
One more suggestion: if you can conduct reaction with gradient and determine the temperatur range for reactions, may be you can combine reactions with for example 58 oC.
Moreover, you can try to use 2 step instead and combine annaling/elongation + detection.
if the RT-PCR machine have the gradient option, then you can create a range to cover all your primers. However, if that option is not available, then you will have to run the primers individually
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