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-   -   Two standards. Two curves. (http://www.molecularstation.com/forum/real-time-pcr-quantitative-pcr-forum/87844-two-standards-two-curves.html)

KaptanOblivious 05-02-2013 04:48 AM

Two standards. Two curves.
 
I'm sorry if the answer to this is something obvious that I'm missing - I am quite new to this and have been trying to figure this out for 2 weeks now.

The Problem:
I am using a standard taqman reaction to amplify viral genomes from dna isolated from mouse tissues (Qiagen DNeasy kit), and am getting very nice and consistent data within experimental groups, standardized to histone control. I am, however, getting very different calculated copies depending on which set of standards I am comparing to. I am amplifying a specific viral gene that has one copy/genome. One set of standards consists of this gene inserted into a small plasmid (2.5kb). The other standard is a Bacterial artificial chromosome (BAC) and contains the entire viral genome (>100kb). The purity of my standard DNA seems OK. However, I get very different results from these standards (1.5-2 logs difference) with the smaller plasmid giving me higher genome copy reads in my samples. The dilutions and calculated copies have been checked and rechecked. The standards both give fantastic curves, but are very far apart. The most obvious answer is that they aren't at the concentration I think they are, or aren't diluted properly, but I have checked and rechecked this. What gives?

luisillo 05-02-2013 03:39 PM

Re: Two standards. Two curves.
 
It is because the plasmid is smaller than the BAC, so in the same DNA concentration you will have a larger quantity of copies of your gene in the plasmid than in the BAC. This will ultimately lead to a larger quantity of copies of your amplicon in the plasmid standard. However, I don't know if this the difference can be as big as you describe (1.5-2 logs), but, as I stated, the plasmid yield will be bigger than the BAC yield.

KaptanOblivious 05-02-2013 08:36 PM

Re: Two standards. Two curves.
 
So I keep thinking that it must be something like this, but I have calculated the copy numbers correctly based on the size of the plasmids being used, both by hand, and using online tools. I'm thinking there may be contaminating DNA in one of my plasmids, but the both look fine cut vs. uncut on an agarose gel.


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