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Male
hii
forward primer - 27nt, Tm-65, GC%-48.1
reverse primer - 28nt, Tm-65.1 GC%-46.4
i have set gradient pcr with annealing temp-60 62 64 and 66
early denaturation- 95 C - 4 min
denaturation - 95- 15s
annealing - 15s
extension- 72- 30s
late extension - 72 - 7 min
35 cycles
expected amplicon size- 1 kb
i did not get any amplification
expert advice needed
