In OE PCR, I have to stitch two gene sequences, so the annealing temperature in the first cycle would depend on the overlap reagion between two products designed with appropriate primers or would depend on the end primers used?
you have to run the pcr reaction many times until you find the right temperature. its labouurinag nd time consuming
Just check for the melting temperature for the annealing component. Run a PCR with the gene products with NO PRIMERS for about 5 cycles or so to anneal the DNA together. I reccomend about 100-500ng of DNA in total for the gene products. This allows the DNA to anneal and for the polymerase ro extend and fill 5'-3' of the now considered overhangs.
Now run add primers in and change the annealing temperature to your primers and run for about 30 cycles.
This would then amplify the DNA as it was previously only extended and no extra gene products were formed.
Make sure to gel purify after this.
Annealing temperature do not depend on the target but on the primers you use. you can test the Tm of the primers in many websites, for human and higher species you can test the in silica PCR tool from the UCSC.
fred
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