I used reported primer sets (first and nested PCR) and followed their protocol.
I'm getting a similar result to the reported result. However, for the nested PCR, I'm getting two bands that are unlikely to reported one. The nested PCR product size is 524 bp only.
I'm considering whether the first PCR primer set (1,180 bp) would affect the multiple bands, or does the annealing temperature needs to be optimized?
In the figure around 524 bp is the target, but 780 bp doesn't know.
I do not think that the annealing temperature is critical for the second nested pcr so long as the primers do anneal the very small length of dna template will mean that the second round primers are very specific. I think that the problem is more likely that you are not diluting the first round product enough. I used 1ul of 1:100-1:1000 diluted first round product. The primers in any pcr are in huge excess so if you are just transferring 1ul of first round pcr mix into your second round pcr than there will be ample outer primers to form amplimers od Fout-Rout, Fout-Rinner, Finner-Rinner and also Finner- Rout so You may just be seeing some of these options depending on the annealing temperature of the second round pcr
I would cut the upper band from the gel, purify it and sequence it.
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