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.
Volumes do not help much Ichini Sudhir . Can you give us the primer sequences,expected pcr size and the concentrations of the pcr reagents and dna that you are using please. PCR buffer often comes as 10x buffer in which case you should be adding 2.5ul as your total volumes add up to 25ul. Does the buffer already contain Mg?
Are you making one "master mix"/"cocktail" of all of the reagents except the DNA & then aliquoting? Adding each reagent individually to each reaction is a recipe for too much variation. And as Paul Rutland says, check your math. You need to worry about concentration of each reagent.
Magnesium precipitates easily too. If you see specks of white in the tube, then you need to toss it and get a new one.
Honestly, I'd suggest buying a ready-to-use PCR mix that contains the buffer, dNTP, magnesium, and enzyme in one tube. You just add primers, template, & enough water to get the final concentration & volume you need.
Amplification of the COI gene using LCO 1490 and HCO 2198 primers have, sometimes shown a relatively low amplification success rate or even failed: Article Are “universal” DNA primers really universal?
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