The forward primer has a tm of about 60ºC and the reverse, which is the Sp6 promoter primer, is merely 45ºC.
Last couple of PCRs where getting better as I lowered the temperature of annealing to 51ºC, but I haven't still got a really nice glowing band.
No access to gradient termocycling. In what way should I mess with MgCl2 Concentrations?
Hi Esteban
I would suggest to run the PCR at the annealing temperature lower than the Tm of Sp6 primer. In this way both primers will anneal. The exact temperature will depend on your Taq polymerase. For example Dream Taq from Thermo works at Ta 5 degrees lower than the Tm of the primers.
Good luck!
Natalia
Check your primer with Tom calculator available in online, different taq polymerase and its master mix will work in different annealing temperature. Check according to your taq and its master mix.
The problem with dropping the Tm so low below the 60°C primer, is non-specific amplification. At 45°C (the bare minimum annealing temperature for any PCR), the primer will anneal non-specifically and potentially generate background bands.
Hello Esteban,
I am facing the same problem with my primers. Did you manage to resolve it and how? Thank you.
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