hello,
i want to know why according to the literature when they establish standard curves they use cloned bacterial DNA (for exemple for feacalibacterium strains) in a plasmid and for other strains for exemple bifidobacterium they extract DNA directly from culture without cloning ? what are the interest to proceed to cloning for some strains and not for others ?
Thank you
Well, i think it's up to the difference between relative quantification and absolute quantification. If you want to know the absolute copy numbers of a gene, you should cloned the DNA.
Both methods will generate absolute quantification provided that you know the concentration of the control target DNA, by spectrophotometry for example, and know the DNAs FW if the read-out will be DNA copy number. There are general reasons for doing one or the other, but for your examples above, there may be some specific answers.
In some cases, the lab has the qPCR target cloned into a plasmid already and the ease of prepping and quantifying a plasmid is very straight forward compared to culturing the organism itself and prepping the chromosomal DNA. Some people may not want the clone in a plasmid because it can more easily contaminate qPCR than the bacterial genome. Lastly, qPCR amplification from the chromosome may be more difficult than from a small plasmid, so the Ct values obtained from x copies of a plasmid may differ from x copies of the chromosome. So plasmid would be an easier standard curve to generate but chromosome may give more accurate genomic copy number values.
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