How can I create standard curves for qPCR

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Tu
Tu's picture
How can I create standard curves for qPCR

This is the first time I work with qPCR and I have to do standard curve for LacZ gene of E.Coli.  I have protocol to create standard curves with genemic DNA or plasmid DNA Templates for use in qPCR.  I wonder I could use this protocol or not. And if not, which protocol should I use. 
Thank you so much

Ivan Delgado
Ivan Delgado's picture
 

 
Hi Tu,
The idea standard curve recreates as much as possible the conditions used to extract the DNA you will be testing. So if you are trying to quantify the amount of DNA (lacZ) present in a particular E. coli sample, then you should extract the DNA containing your LacZ gene standard using the same DNA extraction conditions. For example you could have an E. coli containing the lacZ gene, grow it, extract DNA, and quantify the DNA using a UV spectrophotometer (or DNA quantification kit). Once you had this DNA quantified you can use it as your standard curve. 
You can use your plasmid DNA as a standard, but just keep in mind that the way you extracted that DNA, or the quality of that DNA, may not be the same as the one from your unknown samples you hope to quantify using your standard curve. In that case, your quantification may be off because the DNAs are of different qualities, composition. 
Good luck