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PCR Inhibition by DNA? - (Jun/17/2008 )

I am using plasmid DNA (from a Qiagen midi kit) to generate a standard curve for use with qRT-PCR. My curve consists of a standard five-point serial dilution ranging from 100ng to 0.01ng of DNA in a 25uL reaction volume; all five concentrations were diluted from the same source.

My problem: while the more dilute points (1ng, 0.1ng, and 0.01ng) amplify successfully, I consistently see no amplification in the highest two points (100ng, 10ng).

I do not believe that reagents are limiting because the reaction was optimized using (genomic) DNA at higher concentrations than those in my curve. Therefore I can only assume that something is inhibiting amplification in the 100ng and 10ng samples, and a dilution of two orders of magnitude is sufficient to overcome it. If anyone has other suggestions, please feel free to comment.

A Qiagen representative suggested that my DNA may be inhibiting PCR. While I am not convinced that 100ng of DNA in a 25uL reaction tube is enough to cause problems on its own, is it possible that my plasmid DNA is mechanically inhibiting PCR (e.g supercoiled)? If so, how should I address this?

Any input is much appreciated. Thanks in advance!

-X-Gal-

What elution buffer do you use? The Qiagen EB Buffer? Maybe the salts in it are causing some trouble. Also some residual ethanol from the miniprep may be not that helpful for an efficient PCR reaction.

-jazim-

This is common, and why people should dilute their DNA as a PCR template. The problem is usually not excess DNA, but rather PCR inhibiting contaminants of the DNA. Dilution makes these have little effect. You could try purifying your DNA more, but that is likely not as good a choice as doing your curve with even more dilute DNA.

-phage434-

QUOTE (jazim @ Jun 19 2008, 05:08 AM)
What elution buffer do you use? The Qiagen EB Buffer? Maybe the salts in it are causing some trouble. Also some residual ethanol from the miniprep may be not that helpful for an efficient PCR reaction.



I did use the Qiagen buffer EB to elute. However, the subsequent steps call for precipitation in isopropanol followed by a wash in 70% EtOH. In addition to this, I performed a second precipitation (in EtOH this time) after the initial PCR in hopes of "cleaning up" the sample, but this did not affect the success of my PCR.

-X-Gal-

QUOTE (phage434 @ Jun 20 2008, 02:31 PM)
This is common, and why people should dilute their DNA as a PCR template. The problem is usually not excess DNA, but rather PCR inhibiting contaminants of the DNA. Dilution makes these have little effect. You could try purifying your DNA more, but that is likely not as good a choice as doing your curve with even more dilute DNA.



Unfortunately I experience low DNA yields from the midi-kit I have been using, and have been unable to acquire sufficient DNA to achieve my highest point from a dilution. But thank you for the input.

-X-Gal-

Not to go off topic, but what concentration of template genomic does everyone run to be sure you have an adequate quantity of DNA to feel confident in the reaction. I am aware that different products may have different frequencies in the genome, but what is convention, and how was that derived?

currently I run 250ng/ 25┬Ál rxn volume in a sybr green DNA PCR.

Randy

-Randoramma-