For example we have a bacterium containing two plasmids, one of which carries a target gene. Is it possible to calculate the number of copies of this particular plasmid performing real-time PCR analysis of the target gene?
Thank you!
Yes, that is possible to do. You'll need a standard curve of known copies of the target gene. To adjust for the number of cells present in your DNA extraction, you'll need either a good count of the number of cells for each sample or another standard curve with a gene from the main bacteria genome. You could set up your experiment as either a measure of exact copy number or relative to the main bacteria chromosome.
Hi, thank you for you reply! So I need to perform qPCR with the known amount of the target gene, make a standard curve and then extrapolate the previously obtained results of whole genome DNA qPCR analysis to this stadard curve?
I am unsure of how to do this using qPCR, but if you have sequenced the bacteria genomes you could calculate coverage of the plasmid contigs and compare that to coverage of the chromosome. For example, if you have 10x more coverage for the plasmid you could estimate a copy number of 10 for the plasmid.
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