Dear all,
I am quantifying bacteria via qPCR using SYBR-green assays and targeting the 23S rRNA-gene which is available in multiple copies in bacteria.
My question is how do I accurately calculate the copy number when targeting this gene?
I usually use this formula:
X ng * 6,0221E23 / (Genome size in bp * 660 g/mole) * 1E9 ng/g
Do I have to divide the result I am getting from this calculation by the number of gene copies or do I divide the genome size by the number of the gene copies and add it to the formula?
Kind of having trouble to get behind the right formula. An explanation would be greatly appreciated!
Thanks!
Thank you for your answer Florian.
I am investigating genomic DNA of various Lactobacillus strains and I want to compare CFU vs genome copy number.
I use species-specific primers targeting 23S rRNA. Genome sizes and copies of 23S per genome are known.
I second Florian. You need a standard curve to get the target gene copy number N from the measured Ct value. If you then know that one cell contains k copies of the target gene, the number of cell-equivalents you measured is N/k.
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