I have extracted DNA from Sitophilus oryzae adult using DNeasy Blood and Tissue kit.

I am doing PCR to amplify COX1 using universal primers LCO1490 and HCO2198 I am not getting band. Sometimes bands are there but around 400-500bp

I am using chemicals separately for pcr in the following concentrations

Buffer= 2ul

Mgcl2= 1ul

dntp= 1ul

Forward primer= 1ul

Reverse Primer= 1ul

Taq= 0.5 ul

DD water= 17ul

DNA = 1.5ul

and PCR conditions

94°C---3 min

94°C--0.30

49°C--0.40

72°C-- 1min

36 cycles

72°C---10 min

hold- 4°C

I have tried other combinations, also gradient from 46-54°C and touchdown, still not getting uniform results. I have used negative control also to check whether there is some fault in chemicals but nothing was there. Could you please suggest to me what I should do so I can get uniform and accurate PCR bands.

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