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Absolute quantification


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#1 Ralphie

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Posted 26 November 2009 - 07:27 AM

Hi
I want to identified and quantify bacteria species from biological samples. To quantify the number of an particular specie per g or l of sample, can I do a standard curve with PCR product dilution (using the specific primers used to identified the strain). In the protocols I saw they used plasmid standard curve, and I thing using PCR product will be more easy.
Thanks

#2 BioMiha

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Posted 04 December 2009 - 05:34 AM

Hi
I want to identified and quantify bacteria species from biological samples. To quantify the number of an particular specie per g or l of sample, can I do a standard curve with PCR product dilution (using the specific primers used to identified the strain). In the protocols I saw they used plasmid standard curve, and I thing using PCR product will be more easy.
Thanks



Hi Ralphie,

You can quantify the PCR product with real-time PCR, but with old fashioned PCR it's not going to work.
You can correlate the plasmid quantity to bacterial quantity because the plasmid quantity is determined by its copy number in a bacterial cell, which is more or less constant at least looking at the whole bacterial population. But the PCR product quantity at end point is determined by the PCR reaction conditions and you will get the same amount of product no matter what starting concentration of bacteria you have if the PCR reaction reaches plateau. That's why they came up with real time PCR = to measure PCR product accumulation in real time and not at end-point.
Hope it helps.
Cheers,
Miha




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