I'm currently optimizing my nested PCR to detect Paramyxovirus with 639 bp expected band on the first round and 561 bp expected band on the second round. For the first round, i use forward primer (I) with Tm 61,8°C and reverse primer with Tm 62,5°C. For the second round, i use forward primer (II) with Tm 56,1°C and reverse primer with Tm 62,5°C (the same reverse primer as the first round). What is the appropriate annealing temperature for the first round and second round that i need to try?
P.s. my lab doesn't have gradient PCR, so I'm relying on a thermal cycler that can run a conventional PCR.
You can use the online tool to determine the annealing temperature for the PCR based on your forward primer and reverse primer sequences. You should mention the kind of polymerase, either Taq or high-fidelity polymerase, for the PCR reaction, which will also influence the result. I give the link below for one of such online tools: https://tmcalculator.neb.com/#!/main
For the round 1 primers, annealing should be at 60-62 °C (61° C optimal), but the issue here is round 2 primers have large ΔTm (6.4°C), either go with a test 57–59°C + additives (DMSO, betaine, MgCl₂) to enhance your reactions, but the solution for long term is to redesign a forward forward primer (Tm ≥60°C) via Primer-BLAST.
https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi