# Detection of 10 plasmid copies - detection limit (Jul/29/2007 )

Hello

I'm very new in qPCR and I have a (might simple) question:
Is it possible to detect 10 or even 1 copy of plasmid respectlively 1 cell by SYBR-GREEN assay?

Thanks!

-see9-

i think 10ng are the limit...

-fred_33-

I have experience with these small template numbers. First, the lower the DNA content is, the more your data suffers from statistical deviations. Poisson distribution e.g. says, that by determination of a mean number, the accuracy deviates by the standard deviation represented by the square root of the mean value. If your DNA solution contains statistically 10 copies, then you would measure a mean value of 10 copies +/- sqrt(10) or a relative error of 31 %. This means that to determine even a such high number like 10 copies, you have to do more replica than only one single sample to catch the exact value, since a single measurement will be in 32 % of cases even outside the range of 10 +/- 3.1 copies.
The next problem are numbers smaller than 10 copies. At the moment I'm not so familiar any more with poisson statistics, but I can tell you that the more you approach the zero template number, the higher the statistical error becomes (it's not the standard deviation any more). But you can still measure in this range but even if you use more than three replica, you won't be able to discriminate 5 templates from 3 (you know what I mean?). In practice you will see PCR failures (zero templates) in some of your replicas, since the DNA concentration is so low, that by pipetting a certain volume you don't catch a DNA copy at every try. At this moment some figures lie at my desk. The calculation of a mean two copies value had a standard deviation of +/- 1.77 copies or a relative error of 88.5 % (!). From three replica only two successfully amplified a PCR product. Beneath this calculations you must be aware of systematic errors, regarding to your quantification. Perhaps you think your DNA solution has to contain 50 targets and in reality it is only 1 target. If you don't trust it, you can try to check it by serial dilutions (knowing how low template numbers are affected by poisson distribution) to find the 1 to 10 template range.
hope I could help

-westenmax-

I routinely detect 10 copies, and yes.... because of sampling error you can get some variation in results but I've had good luck.

I've also found that when working with small copy number samples, it is more helpful to make serial 2X dilutions instead of serial 10X dilutions. You will have much more confidence in the results, when you see them dropping by one cycle for each dilution.

I don't usually go below 10 copies on my standard curve, but if I wanted to I would go to 5 copies, then 2.5 copies, then 1.25 copies.

-Patty4150-

it is possible ..even i standarised the condition where i can pick 10 copies comfortably. But this all depends on our gene and pcr condition. if u want to achieve u have to standarise in all prospective like magnesium,primer,anealing temp, cycle number,etc., if u optimise all this it is possible to pick even less..but u have to put lot of effort to get it.

A.Avinash

"I never did anything worth doing by accident, nor did any of my inventions come by accident; they came by work"-Thomas Alva Edison

-Appuu-

QUOTE (Appuu @ Aug 27 2007, 07:34 PM)
it is possible ..even i standarised the condition where i can pick 10 copies comfortably. But this all depends on our gene and pcr condition. if u want to achieve u have to standarise in all prospective like magnesium,primer,anealing temp, cycle number,etc., if u optimise all this it is possible to pick even less..but u have to put lot of effort to get it.

A.Avinash

"I never did anything worth doing by accident, nor did any of my inventions come by accident; they came by work"-Thomas Alva Edison