Template DNA (plasmid) concentration for PCR - (Nov/15/2011 )

What concentration of template plasmid DNA (pET, 6kb size) is the best to amplify a 0.8kb fragment from it? I used a 3.5 ng/25ul and didnt get any amplification. Will the problem be its supercoiled form? Would cutting with a single enzyme help? Any similar experiences?

go atleast 20 to 30 ng of plasmid....

That's not your problem. PCR should be able to amplify (in principle) single molecules, so even a picogram of DNA is easily within reach. The usual problem is primer design, or leaving out a key component of the mix.

Thank you Gnana for the suggestion. I dont have many options when designing the primers since I have to design them from the 5' end and 3' end of a gene. Have to try changing other PCR conditions.

To have an equivalent of 100ng gDNA which is a PCR standard template concentration, you should use 0.2ng of your plasmid. Usually very small amounts of plasmid are required, because there are lots of copies. Using too much of a plasmid can lead to inhibition of the reaction and you would see no product. Try to dillute it more.