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qPCR std curve calculation issue if using PCR product as the std

qPCR calculation

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#1 Garth Owen Watson

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Posted 08 March 2016 - 08:51 PM

To prepare for a set of serial dilutions of qPCR standards I follow an online protocol that assumes using either gDNA or a plasmid. You then identify the mass of DNA per genome and divide by copy number and away you go with the subsequent calculations. But my problem is this....if I were to use synthetic standards ie; custom oligos that are surrogates for the PCR product......they are short in length and therefore do not relate at all to the genome of interest in terms of "mass of DNA" .....is  this a problem and if so is there an alternative protocol for the calculations? I am using the Applied Biosystems online protocol presently.

 

I guess my question even extends to using a plasmid with the PCR product inserted as it bears no resemblance in mass to the genome from whence it came. It has bugged me for sometime.



#2 Trof

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Posted 14 March 2016 - 03:04 PM

AFAIK you never know the copy number, and that's what you need to get for qPCR standard.

 

What you need is DNA concentration to copy number calculator, like this one for example (for the type of template you have, but I suppose dsDNA). You need concentratration that you measure (fluorimeter measurement is more precise, but you can go with spectrophotometer as well) and length of whatever you have, from plasmid, to oligo, to PCR product, given that it's pure.

 

When you get a number of copies, you need to dilute to roughly a copy number equivalent of your sample (or rangle around it) I use a golden standard 100 ng of human gDNA to reaction which is in 104 range for single copy gene (you can use the calculator to calculate that too), but that is in a complex template, the less complex template you have the more efficient PCR is going to be (and Cts lower) in the same copy number.


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