PCR Inhibition by DNA

[Dionisia]

HELLO ALL!

I have a big pcr problem. I am trying to conduct a pcr using plasmid DNA I purified using a Qiagen midi kit.

I am using this DNA to make a standard curve for qRT-PCR.

My standard curve for the qRT-PCR consists of a five-point serial dilution which ranges from 10ng to 0.01ng of DNA in a 25uL reaction volume.

My problem is that i see no amplification with the highest two points on the curve being the 10ng and 1ng spots.

I dont think my reagents are limiting because I optimized the PCR reaction at higher concentrations than the ones on my curve. Something is inhibiting my PCR amplification in higher DNA concentrations and even a Qiagen representative suggested that my DNA may be inhibiting PCR.
Does anyone have any ideas?
thanks for everyones help in advance
cheers

[postdock]

Hey there,

what elution buffer did you use, the Qiagen EB buffer?

There are salts and EDTA in Qiagen EB that may be causing the PCR trouble.

Also dont forget that there could be residual ethanol from your miniprep that may inhibit the efficiency of the PCR reaction.