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Thursday, May 5, 2011

Copy number calculation for QPCR

A serial dilution of linearized plasmid DNA is used to generate a standard curve for QPCR.  Knowing the size of the plasmid that contains the gene of interest one can calculate the number of grams/molecule also known as copy number as follows:

Weight in Daltons (g/mol) = (bp size of plasmid+insert)(330 Da X 2 nucleotide/bp)

Ex. g/mol=(5950 bp)(330 Da X 2 nucleotide/bp)= 3927000 g/mol

Hence: (g/mol)/Avogadro's number 6.02214199 × 10^23) = g/molecule = copy number

Ex. 3927000g/mol/Avogadro's number 6.02214199 x 10^23) = 6.52 x 10^-18 g/molecule.  

Knowing the copy number for a plasmid and the concentration of the plasmid that is added to each PCR reaction, the precise number of molecule in that reaction can be determined as follows:

Concentration of plasmid (g/µl)/copy number
Ex. (3 x 10^-7 g/µl) / (6.52 x 10^-18 grams/molecules) = 4.6 x 10^10 molecules/µl

Having calculated the number of molecules in a µl of linearized plasmid solution, a series of dilutions can be made for subsequent amplification allowing one to generate a standard curve.  For the standard curve, the copy number of the unknown samples can then be derived.

More on generating standard curve and calculating amplification efficiency

Sourcehttp://www.mncf.tulane.edu/Protocols/copy%20number%20calculation%20for%20qpcr.pdf