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.