I’ve been spending more time in the lab recently, which has allowed me to do some hands-on things that I previously had to try to advise people on without ever having done. This includes something as mundane as using the Qubit dsDNA broad range kit to quantify some plasmid DNA prep concentrations. Here are some notes for people in my lab to keep in mind in the future.

1. Standard 1 is just buffer. And it essentially gives the same amount of background signal, regardless of the volume of buffer or working solution (WS) added (within reason). For example:
   • 10uL of S1 in 190uL WS: 41.55 RFU
   • 10uL of S1 in 90uL WS: 44.72 RFU
   • 5uL of S1 in 95uL WS: 45.32 RFU
     
2. The RFU (relative fluorescence units) that are reported are based on DNA concentration, rather than total DNA input.
   For example, with the S2 standard (at 100 ng/uL):
   • 10uL of S2 in 190uL WS = 1000 ng total, 5 ug/uL = 2421.87 RFU
   • 10uL of S2 in 90uL WS = 1000 ng total, 10ng/uL = 4914.62 RFU
   • 5uL of S2 in 95uL WS = 500 ng total, 5 ug/uL = 2546.59 RFU
     
3. 200uL for a final volume in the tube is overkill. Looking at those standards, we get nearly the same exact RFU numbers when scaled down to 100uL. I mostly view this as ThermoFisher wanting you to burn through your reagent faster so you spend more $$$.
   
4. I don’t have any data on hand to show this, but I swear that a year or two ago, somebody in my lab showed that you don’t have to use ThermoFisher branded Qubit tubes to actually use the qubit. I don’t quite remember what they used (whether it was 200uL PCR tubes or some off-brand 0.5mL tubes more akin to the Qubit tubes). This makes perfect sense, since all it needs to do is allow light to pass through. So ya, I imagine that most clear tubes are fine; you’ll of course want to make sure you’re using the same tubes for both the standards calibration and reading your own unknown samples.
   
5. If you do want to scale down your WS volumes to conserve reagent, it gets a little tricky since the Qubit assumes you’re doing the standards calibration step as recommended by ThermoFisher (ie. for S2, it assumes that you’re doing 10uL of S2 in 190uL of WS, for a 20x dilution of 100 ng/uL to yield a in-tube concentration of 5ng/uL). So while it lets you adjust the volume of your sample you’re putting in (ie. you can tell it a volume between 1uL and 20uL), it doesn’t let you do the same thing for the standards, even if the concentrations end up being the same (like in the 5uL S2 in 95 uL WS case). If doing the 5:95 route, I suggest doing the same thing for both the standards and your samples, and just “telling” the Qubit machine that you’re doing 10uL. Then, if you need to do a smaller amount b/c the DNA is too concentrated (eg. 2uL instead of 5uL), then apply that dilution factor to the “told volume” (eg. 4uL assuming you told it 10uL).
6. Probably the most important one: I saw a pretty huge discrepancy between what the Spec told me (measured with a Take3 plate on the BioTek) and what the Qubit was saying. For most samples, it was roughly a 2-3 fold diff (eg. 53ng/uL by spec, 24 ng/uL by Qubit), although there were also some samples that differed by 4-5fold (eg. 57 ug/uL by spec, 12.8 ug/uL by Qubit). I have no clue why this discrepancy exists……..some of it can probably be explained by contaminants affecting the spec readings, but definitely doesn’t explain the whole thing,