Tm value of one of my primer is 52.8 (50mM NaCl) and another one is 54.5 (50mM NaCl) as given by the primer making company.
Primer Sequence is – i> 5' TGT GTA TCA TAG ATT GAT GCT TTT GA 3' and ii> 5' AAA TAA AGA TGT CAG ATA CCA CAG CA 3'. But when I calculate by manually or by online it shows different. What is the Exact Tm Value? Which one is correct?
What will be the annealing temperature of my PCR reaction? My PCR product size is 282 and GC% is 34.