Hello to all,
I need to perform a standard curve for metagenomic analysis with qPCR, of Treponema Denticola and Pseudoramibacter Alactolyticous, using the 16S RNA copies of my DNA.
As well I must perform a standard of a monk microbial community purchased by the Zymobiomics.
Since it is the very first time that I come across this topic I am sicking for help so I asked help from another colleague and she has helped me a lot BUT, in her calculations of 16S RNA copies and bacterial population she has used the guide of applied biosystems where the Molecular wight of DNA is reported to be 660 g/mole and according her calculations the DNA mass is equal to 9,13*10^20 bp/ng.
I asked the technical team of Zymo and they replied that I should use the following formula 6,022 x 10^23/10^9/650 which equals to 9,26462E+11 bp/ng. At this point I am totally stuck and I can not proceed with my calculations.
According to your experience could you please help me?? Should I consider as the correct molecular weight of the double stranded DNA the 650 or the 660???
Why I obtain 9,26462E+11 and my colleague 9,13*10^20??
Thank you
You are confused because you are just seeing the values and not thinking about them. Both could be correct but both are not the same thing. One is the result of calculation and other is a part of formula to be used.
Check this before you proceed
https://www.researchgate.net/post/How_to_convert_ng_ul_of_DNA_concentration_to_pmol_ul
The molecular weight of DNA can vary depending on the method of isolation, purification, and the type of DNA (e.g. single stranded vs double stranded). Therefore, it's possible that different sources may report slightly different values for the molecular weight of DNA.
In your case, the Zymo technical team provided you with a value of 9.26462E+11 bp/ng based on a molecular weight of 650 g/mole for double-stranded DNA. This value is consistent with the formula they provided, which takes into account Avogadro's number (6.022 x 10^23) and the conversion of grams to nanograms (10^9).
On the other hand, your colleague may have used a different value for the molecular weight of DNA (660 g/mole), which could result in a different calculation for the number of 16S RNA copies and bacterial population.
To ensure accuracy and consistency in your calculations, it's best to use a single, reliable source for the molecular weight of DNA and to double-check your calculations using appropriate formulas and conversion factors. It's also a good idea to use appropriate controls and replicate samples to minimize variability in your results.
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