How can I determinate the number of copy of a gene when I use the Rotor - Gene Q for qPCR.
You need to have a known genome (non transformed plant, for example) (copy number of your interested gene in known genome and genome length for calculation of your interested gene copy number in each DNA amount for example you have one copy number in genome and length of you genome is 3 millions bp and you have 10 genomes at 1 nano gram DNA). At first you must start real time-PCR with equal DNA for each tube (1 nano gram )(known and unknown genome). After real time PCR you will have the CT then you can convert number of your interested gene in known genome (non transformed plant for example) to CT. Finally, you will know that if CT is 30 in known genome for example, you have 10 copies and the CT of unknown genome is 28 then you have 40 copies.
I hope be helpful.
Determining the exact number of mRNA or DNA copies using PCR or qPCR method is called absolute quantification. This is rather a very simple method were you generate a standard curve with a known quantity of your gene of interest and determine the copies of the target in an unknown sample. You could determine the number copies using the formula
Number of copies = ( amount of dsDNA in nanograms * 6.022x10^23) / (length of dsDNA in basepairs * 1x10^9 * 650)
650 is dsDNA or 330 is for ssDNA
For a start you could read this article
http://www.sciencedirect.com/science/article/pii/S0022175903002230
or you may want to try digital PCR wich would give you an absolute copy number. Unfortunately this wont be available on the Rotor gene. You'd need a sprecific instrument (i.e. RainDance)
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