I am doing an allele-specific PCR to detect a point mutation in the DNA of Aedes aegypti, when I used a tm temperature of 55 °C, I observed some PCR products in negative control, and some nonspecific bands in the samples and positive controls. When the tm temperature was increased to 65 °C, the negative control was clean, positive control without unspecified bands, but, no amplicons were observed in any sample, what should I do?
Are you sure that the product at 55c is not just primer dimer?
Can we see a picture of the gel image. I would run a temperature gradient from 55 to 65 using a dna sample known to amplify with any other primer set to find a temperature that works as expected
This may be due to many possibilities:
May be DNA concentration is low. Try to measure. If less than 10 ng/ul. then need to repeat extraction or use a highly sensitive polymerase.
If you are using Taq polymerase try to change to Phusion or platinum polymerase
If the problem in annealing, you can make also gradient PCR or initial 10 cycles with high annealing then remaining cycles with low one
Try also to be sure that the primers are conserved against different sequences from your target.
@paul we are not sure about primer dimer, but when we run the PCR at 55C, the negative control looks like contaminated, so, it is an option!
@Ahmed, the DNA is up to 15 ng/uL, about Taq, we are using a Green Master Mix from promega, so maybe we should try with Platinum, about gradient, we already did it, and at 63C, controls are ok, but samples doesn't amplify, we are gonna try with 10 cycles with high annealing as you suggested, thanks!
Is the PCR mastermix mixture between the one that you have done in 55 degree is different from the 65 degree? If yes, I think the presence of the band on your negative control at the 55 degree might due to the contamination while you conducting the PCR reagents. To make sure you got the bands on your samples, I suggest to optimise the protocol by doing the gradient annealing temperature and also optimise the primer concentration ratios.
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