I did PCR reaction with PCR mix (from KAPA2G Fast ReadyMix. I usually use this product with no issues), however the result is not good enough. I got heavy low non specific band and probably primer dimer. Tm of both primer is 52. First, I did with annealing temperature of 53. I got desire band product and also low non specific band and primer dimer. Second time, I run my PCR with annealing 50, I did not get the desire product, only heavy low non specific band. I did again with annealing of 63 and the product is available but the smear also remains. I did the PCR using supplier protocol as well. Note: in reference, annealing temperature used is 65. Could you please help me to overcome this issues?
Try a touchdown thermal cycle protocol. You will be making the most possible specific product with the highest highest temp possible. Then once the beginnings of the product are made the stringency will be decreased as the temperature does during touchdown. Also primers need to be specific to your target and not hit elsewhere in whatever genome you are working with.
Primers I used are commonly enough to detect IVS1 5 (G>C) mutation in hemoglobin beta gene.
My PI tells us to accurately measure the concentration of your primers and make sure you add exactly equimolar amounts of each primer to avoid primer dimers.
As my openion, some times when the primer do not aneal if you put high anealing tem. since they do not aneal they also have a chance to come as a smear may be bcause upto some level of addition of nt bases to them by Taq polymerase
Enter your primer sequence in any of online primer design software to see their qualities.
1-use Oligo software for check the character and analysis your primer.
2-Change 3prim of one primer(according to template, you can long forward OR revers primer)
3-you can reduce quantity of your primer in reaction
good luck
I agree with Daniel, you should try a touchdown thermal cycle protocol. Maybe a 65ºC to 55ºC.
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