Im designing four different PCR protocol/program for the first time for a cloning project and I could use some help. My issue is with the melting temperatures for the oligos I have; some of them are very high. For one reaction, I have a 78.2* and 92.3* Tm for my primers and another reaction has 92.2*, 88.3* and 78.2* (I am combining sequences in this rxn)
I dont know how to choose a correct annealing temperature for these two reactions. I know the standard way is denature-1' @94*::anneal- 1' @Tm-5*::Extension 3' @72*.
Are these oligos with the really high Tm useful? How can I go about choosing a good annealing temperature? I have only a few weeks left in this project so I cannot start over
Please help! I would love any advice you can give and will answer any questions that I can.
P.S. I should note that the two oligos in the 90s have tails that I am using to attach a new shortish sequence.