I come from a background in inorganic chemistry, primarily dealing with metal ions in aqueous solutions. I now work for a large molecular diagnostics company and another individual on my team is developing an automated DNA quantitation protocol.
As a (relative) newbie to this industry, I find it surprising that lambda DNA at ~300 - 500 ng/uL and ~48000 bp is used across so many applications, including quantification of NGS libraries at ~ 300 bp. One of the first questions I asked upon joining my team pertained to the use of such a large molecule as a reference standard to measure unknown concentrations such a small molecule for normalization.
Perhaps there is minimal difference between quantification of different DNA sizes, but I was never given a proper explanation for why this might be the case.
Thinking back to my time in chemistry labs, different metal standards with different matrices/complexes were very easy to acquire as NIST certified standards at just about any concentration you could want. Obviously, molecular bio is a world away, but my brain still draws these types of comparisons.
Are there any other DNA standards out there that have wide adoption?
If not, does anyone know where one could at least find dilutions of lambda at lower concentrations than what is commonly found? e.g. 1 - 50 ng/uL, certified?
Apologies if these questions may be vague or poorly worded. Any insight or useful discussion is thoroughly appreciated.
Ive done dna quantification using qubit buffer on a plate reader and used the dna standards they shipped with the buffer. We got pretty good results this way and integration with the plate reader + robot means you can do fully automated sample normalization.
I can help with why there isn’t a different in quantification between different sizes of DNA with fluorescence. When you do quantification with lets say a Qubit you stain the DNA with the fluorescent die, and it binds some amount of dye per BP on the DNA, so a 10kbp strand might have lets say 1000 bound dyes, and a 100bp strand 10.
When mass is the output value this ends up resulting in the same measurement since a strand x times larger than another also has x times the mass. Where base pair length does matter a lot is when you then have to change that mass measurement into a molarity measurement, which then means you do have to correct for length of DNA. 10 ng of a 10kbp library vs a 100bp one would have a 100 fold difference in molarity.
If you are looking for a standard set I would recommend this one from Biotium, they also make some great really low level quantification kits if you wanna do something like ct/cfDNA quant with ultra low volumes.
The buffers are identical (or at least interchangeable) but the standards in the Quant-it kits have finer granularity. This is helpful for making sure you’re in the linear range of the plate reader.
