thanks Natalie. i saw the site earlier. However, I am attempting to substitute 7 amino acids (21 nucleotides) in the middle of a gene. So in my case I have a 54 nt primer, whose calculated Tm acc. to IDT is 73, whose two arms have Tms of 34 and 54. However, since the region between these two arms also has 21 nt with matches and mismatches for the amino acid substitutions, how do i calculate the Tm? I'm thinking of setting up a gradient for the annealing temperatures, but do i also need to change other parameters like annealing time to account for size of the primer? Also is the PCR product of a single primer SDM PCR visible on a gel? or do you use the PCR product directly for transformation post Dpn1 treatment? Also the primer is phosphorylated at the 5'end

I'm not sure I understand the question. If you have cloning primers for a bit of DNA that you've since cloned into a plasmid and one primer designed to mutate 21 base pairs, you could use a megaprimer procedure where you amplify ~half the gene using the mutagenesis primer and one of the cloning primers. Gel purify this, and use this PCR product as one huge primer with the other cloning primer in a second PCR reaction using the original plasmid as template. I don't have good recommendations for the annealing temperature for the second, megaprimer PCR, but a gradient going to high temperature should work.

Otherwise, I previously posted about using overlap extension PCR for someone elses SDM question. This procedure is more robust compared to Quikchange (what I believe you're doing) and the above megaprimer reaction:

"Using a second, overlap extension PCR, you can make whatever changes you want because your primers only need to anneal at the 3' end in the first PCR reaction.

In short, 3 PCR reactions are used to first generate your sequence in two halves and then to join the two products together. The first step involves mixing-matching your 4 primers into 2 reactions to amplify your halves. The primers consist of outside primers which would amplify the entire region of interest and mutagenesis primers that anneal within your sequence of interest but on either side of the site to be mutated. You can create whatever sequence you want between the sites that these mutagenesis primers anneal to your template as long as the two, mutagenesis, 5'-ends are the reverse complement of one another. These 2 "first reactions" are spiked into a third reaction, given a few cycles without added primer to extend the full length sequence, then outside primers are added back into the mix to amplify your full, mutant sequence (gel purify here for sure). This piece now has to be cloned back into your vector (use the infusion kit, and this'll be trivial)."

You can try Kunkle mutation.

Thanks guys