Hi All,
I'm wondering if anyone has used the methods on page 51 of the ddPCR Bulletin 6407 from Bio-Rad to determine the total copy number of a gene of interest in a known starting mass of DNA which relates directly to a cell number.
I am not looking at RNA, the sample is extracted gDNA from a known haploid cell population where the gene copy number per cell is 1.
e.g. My gene of interest has 8 copies per uL in a starting ddPCR reaction mix of 22 uL and I loaded 5 uL of diluted DNA into the same reaction. I therefore have 35.2 copies/uL in my diluted DNA sub-sample.
My sample was diluted to a specific concentration in order for me to perform the reaction, so I multiply my 35.2 copies/uL in the by the volume of the diluted DNA (e.g. 100 uL) and then by a dilution factor (e.g. 5) which shows how many aliquots of the same volume and diluted concentration can be made from my original sample. So my total copies in the original sample would be 17 600. Which means that 17 600 of my cells that I used to create my DNA have the gene of interest.
Has anyone done this much backwards extrapolation?
If anyone has used the methods described in the bulletin/exampled above and has published their findings, please could you send me a link to your paper?
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