A review of the Qubit fluorometer for quantifying DNA, RNA, and proteins (page 2 of 2)

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A review of the Qubit fluorometer for quantifying DNA, RNA, and proteins

(page 2 of 2)

The Quant-iT dyes are also quite insensitive to the presence of salts and other contaminants. I was never really able to accurately measure the amount of DNA remaining from my gel purifications with a spectrophotometer, but the Qubit can measure them just fine. With the Qubit, I've even been able to quantify extremely low concentration DNA samples like ChIP DNA for the first time.

Negative features of the Qubit and Quant-iT assays

Setting up a Qubit based quantification is slightly slower than using a spectrophotometer like the Nanodrop, because you have to prepare the dye and buffer for each sample, you have to prepare two standards for each set of samples, and you have to place each sample and standard in its own tube for quantification. ~~In addition, the Quant-iT dyes come as a 200x solution in DMSO which is stored frozen at 4C~~ (DMSO melting point is 18.5C) ~~and takes quite a while to melt at room temperature; so it takes a little extra planning to make sure the dye is ready for your experiment.~~ Because of the extra time and cost for running the Qubit when compared with the Nanodrop spectrophotometer, I use the Nanodrop for pure samples (i.e. containing only RNA or DNA and not a mix of the two) above 30 ng/ul.

Another negative aspect of the Qubit is that it requires a lot of sample if the sample has an extremely low concentration. You prepare the quantification reaction with 1-20 ul of sample. For high concentration samples 1-2 ul is fine, but for extremely low concentration samples it is necessary to use 10-20 ul to have enough nucleic acid or protein to be detected by the fluorometer. Unfortunately, samples are typically extremely low concentrated when you don't have much of them to begin with, so to quantify them with the Qubit you often need to use almost the whole sample. For example, to quantify my ChIP DNA I must quantify 10 ul of a 30 ul sample to estimate the total amount of DNA in the sample. But at least it is possible to quantify it.

My least favorite aspect of the Qubit is that you need to have at least a crude guess of your sample concentration to know which dye to use and to know what amount of sample to use in the quantification – otherwise you'll go outside the bounds of the machine. For example, if 1 ul of a high concentrated sample contains more DNA per microliter than the high standard for calibration, you'll get an out-of-range error. When your sample is above the range, you can either dilute it or quantify the sample with a spectrophotometer.

Update

It turns out my earlier statement that you need to thaw the dye each time you use it was not correct. I received the following info in an email from Jill Hendrickson, the Qubit Project Manager at Invitrogen:

"I also wanted you to know that we did design the kits so that you can store the dye and buffer at room temperature. That's because we knew it would be a pain to thaw the dye out. You just need to store the dye in the dark, like in a drawer."

After digging around my qubit tubes a bit, I did see that although the large container the kit comes in says 4C the individual tubes have their own storage condition labels, and the dye label says to store at less than or equal to 25C (room temperature). This prevents having to plan your experiments around the melting time of the dye and makes the Qubit much easier to use.

Conclusions

Click image for larger

The most important aspect of wetlab experiments in general and novel experimental technique development in particular is knowing what you have in your sample and how much you have of it. Since purchasing the Qubit, I've found multiple occasions where I previously had to guesstimate the amount of RNA or DNA in my sample but with the Qubit I could accurately and quickly quantify my sample. For the bulk of my wetlab work which involves high concentration RNA or DNA samples that are relatively pure, I prefer to use the Nanodrop spectrophotometer which is faster to set up and has no per-sample reagent cost. But for those cases where a sample isn't pure enough or concentrated enough, the Qubit has become an essential part of my molecular biology toolkit.

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