Copy Number Determination with Real Time PCR

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Tony Rook
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Copy Number Determination with Real Time PCR

I am developing an assay to detemine the copy number of a bacterial plasmid. Does anyone have a good reference for this methodology?

trook

SanDiablo
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Do you mean you want to

Do you mean you want to determine what the copy number of your plasmid is per bacterial culture, transfected cell, or just the copy number of a purified plasmid?

Generally, the copy number of a purified plasmid is calculated from taking an OD measurement and determining concentration (sorry, I don't have the formula in front of me, but it involves Avogadro's Number.)

Once you know the #copies/ul of your purified standard, you create a standard curve ranging from ~ 10 copies/reaction to 10^7 copies/reaction, then you can determine copy number of your unknown samples. (Be sure to toss out your non-linear points.) If you are trying to measure directly from bacteria or cells, you can probably just add a few dilutions directly to your PCR reactions.

Good luck.