Dear experts,

I am trying to set up my annealing protocol to make dsDNA out of 2 ssDNA oligos (29 nucleotides each).

I have both oligos in the same 100 uM concentrations and use 40 ul of each. Having equal molar amounts (4nmoles). I use a standard protocol in which you incubate for 5 min at 95C and gradually let them cool off to room temp.

I still do not get an efficient annealing of both primers. Does anyone have tips or tricks for me to get an almost 100% annealing.

Thank you so much for yr advise!