efficiency about 120% ?!? How? and WHY? - (Feb/28/2008 )

How does it come that the slope of the standard curve is more than 100% when I mix 2 DNA??

First let me explain what I am doing right now! I try to quantify a DNA which is mixed within another DNA. THe DNA I need to quantify is the one with the lower amount in the mixture. So I designed a standard curve with mixed samples because the efficiency of the reaction differ a little from those with natural (single) DNA.
How does it come that the slope differs in a direction which is in practice not possible? Means that the slope is around -2.8 which means the reaction has a theoretical efficiency of 127%! Thats not possible, what could be the reason? By the way, the reaction has efficiency around 100% with natural DNA (not mixed!).

And the other thing, what can inhibit my reaction so that the efficiency collapse to 50%? Using other probes than these for the reaction causes porblems described above, the slope sink to -5.3 or sth like this. Its fine by me if the reaction doesn't show an efficiency of 1 but at least 0.9 should be possible. There are those curious slope values when I analyze the mixed samples, with straight DNA everything is normal...

Waaaahhhh... HeLP pleeeease!

Wen you mix 2 templates of course you are introducing a new parameter to your reaction. Can you upload your file becoz I think no one knows what you are talking about. And please describe more detail your protocols

Chris

Here's and answer to the question of efficiency over 100%. It is from an article called 'How Reliable is Your qPCR Data?' from dddmag.com:

"When you're getting efficiency numbers greater than 100%, that usually indicates a saturation of the RT or the PCR, and you see less of a cycle threshold (Ct) difference between higher dilutions. That can sometimes throw off the slope on that high end and create artificially high efficiency. It is very important when you are looking at your standard curves to look for even spacing between all of them . If you do see compression on the upper end, throw out those points and recalculate the efficiency."