Hello i am trying a pair of new primers. Unfortunately I do not have enough reactions to perform a temperature gradient.
My HK primer has Tm of 60.3 (forward) and 60.8 (reverse) and the other primer I will use has 60.4 (forward) and 58.9 (reverse).
However it is also stated in the data sheet that my primer with 60.4 Tm has a strong secondary structure formation. I have seen that even though standard PCR kits have some amount of DMSO it still can be useful to add DMSO in order to reduce the Tm.
If so would you recommend additional DMSO and is 5% is a good starting point?
Also in this case can I blindly predict my Ta by subtracting 5 degrees from the lowest Tm I have?
Any help would very much be appreciated. :)
Best Regards