Between Qubit vs Nanodrop, Nanodrop always wins the race against Qubit. However, Qubit has its own importance in comparison with Nanodrop light.
Quantifying DNA is yet another important step in genomic research. The purity and quantity of DNA influence the result of an experiment. The impurities such as protein, phenol and other salt traces may terminate a PCR reaction. Ultimately it stops the reaction.
Pure DNA has a 260/280 ratio of ~1.80. We had discussed the importance of DNA purity in our previous article on the review of Qiagen Qiaxpert. Before understanding the principle and working for each, we have to understand several basics.
The purity of the DNA can be measured spectrophotometrically. Once the light of the desired wavelength passes through the sample, it will detect the impurities present in DNA. For example, the protein can absorb a 180nm wavelength of light.
The same mechanism is applicable for DNA which absorbs light at 260nm. Passing light of 280nm and 260nm simultaneously from the DNA sample can detect the DNA present in a sample. There are two main techniques to check the quantity of DNA- fluorometry and spectrometry.
Put simply, in fluorometry, fluorochromes bind to DNA and emit fluorescent which a detector detects. Contrary, in spectrometry lights of two different wavelengths, passes from the DNA. Technically, both instruments work on different principles. So in this article, we will go through a detailed comparison between both instruments. We will review both products as well.
We will also discuss the chemistry and principle of both instruments as in the last segment of this article, I will give you my ultimate guide to use which instrument.
The opinion about the instruments is based on our working experience. It is not paid, again the purpose of this review is to provide a guideline for those who want to buy it in the future. Let’s start the topic with basic information.
The name itself suggests that the principle of the Qubit fluorometer is based on the chemistry of fluorochromes. The fluorescent dye is added to the DNA solution which binds with it.
Based upon the fluorescence difference between bound and unbound fluoro-molecules the DNA is quantified. As shown in the figure, the instruments have a digital display, a sample chamber and a USB output.
The Dye stacks with the bases of DNA and emits fluorescence which is detected by the fluorometer. Different types of dye are used for DNA, protein and RNA. It can accurately measure the amount of DNA.
The Qubit is the registered trademark product of Invitrogen (a brand of Thermo Fisher Scientific).
Principle of Qubit fluorometer:
The Qubit setup is very simple and easy to operate. But we have to prepare a sample each time while performing quantification. The Qubit Kit contains a working Qubit Fluorometer buffer, DNA dye and a standard.
Generally, a 20μL reaction is most preferable, however, the company advises performing 200μL of reaction.
Add 1μL to 10μL of a sample to the microcentrifuge vial and add the DNA dye in the same proportion. Next, add the Qubit buffer solution (180μL for 200μL to reaction).
Incubate the sample for 2 to 3 minutes at room temperature and insert the tube into the Qubit Fluorometer. Always run standard (given in the kit) before using the sample. For the preparation of the standard, run only the standard without a DNA sample.
Advantages of Qubit Fluorometer:
- It is the most accurate and cost-effective
- The instrument is smaller and easy to use.
- It accurately quantifies DNA. The instrument is based on fluorescent chemistry hence the dye can bind with the DNA only, so even if the sample is contaminated, it can give the exact concentration of DNA.
- It will give the result within 2 minutes.
- It quantifies DNA as low as 10pg to 200ng.
- The sampling requirement is lower (ranging from 1μL to 10μL)
- Highly recommended for sequencing, microarray and RT PCR
Disadvantages of Qubit Fluorometer:
- Though it can process samples within two minutes, it takes more time than Nanodrop light.
- It required additional sample preparation. We have to prepare the sample for quantification, therefore, it is a bit time-consuming and complex process.
- The sample volume requirement is higher in comparison to nanodrop light.
- The principle is based on the fluorochromes, therefore, it may be hazardous for health.
- The shelf life of the chemicals is also lower.
- Cost-effective, though, additional kit of reagent is always required which makes it more costly.
- It can not measure the quality of DNA.
Nanodrop light spectrophotometer:
The principle of the Nanodrop light spectrophotometer is based on spectroscopy. Here no additional chemicals are used in the application.
The light beam of 260nm, when passes through the DNA, it measures the amount of DNA molecule present in the sample. The principle is based on the absorbance spectra of the sample.
The instrument is manufactured by Thermo Fisher scientific. Thermo fisher scientific is the leading manufacturer in the life science field. They are known for their single-use instruments (no additional utilities required).
Principle of Nanodrop light spectrophotometer:
The working principle of Nanodrop is simple than any other quantification machine available in the market.
Just put a 1μL or 0.5μL of a sample on the stage of Nanodrop and allow it to measure. It will give a result within 2 seconds. However, before quantifying the sample always run a blank with TE buffer or D/W.
No additional utility or chemical is required.
Advantages of Nanodrop light spectrophotometer:
- The instrument is compact and easy to use as compared with any other instrument available in these categories.
- Highly accurate
- The quality and quantity of DNA both can be detected.
- The sampling requirement is as lower as 0.5μL
- It can be used for protein, DNA and RNA quantification.
- It is safe to use (No harmful chemicals are used)
- Additional sample preparation steps are not required.
Disadvantages of Nanodrop light spectrophotometer:
- Sometimes it measures the free ssDNA and some smaller dsDNA fragments hence the accuracy is a bit lower.
- costly than Qubit
Read further on PCR,
A comparative review between Qubit vs Nanodrop:
Qubit is highly recommended between Qubit vs Nanodrop in downstream applications such as sequencing, microarray and RT PCR because these experiments are highly sensitive, a picogram of quantity fluctuation leads to negative results.
However, an additional instrument for qualitative analysis is required along with Qubit because the quality of the sample also matters in sequencing and microassay.
In contrast, the Nanodrop light is highly recommended between Qubit vs Nanodrop for routine PCR and restriction digestion studies because it is faster and measures the quality and quantity both in a single assay.
My ultimate guide for using Qubit vs Nanodrop light
While handling reagents of Qubit always wear glowers. As the company recommended, do not open the lead of the Qubit in sunlight. Do not place the reagents in sunlight as well.
If you want to purchase a Qubit, and worried about the quality of DNA, I have a solution for it. However, for doing this you have to achieve expertise in the analysis of the result.
The cost of the Qubit is much lower than Nanodrop light, so purchase Qubit for your Lab. For qualitative analysis, you can run an agarose gel electrophoresis for genomic DNA. The band type will give you a rough idea about the purity of DNA.
Read further on agarose gel electrophoresis,
- Role of EtBr in electrophoresis
- DNA gel loading dye
- Agarose gel electrophoresis buffer
- Agarose gel electrophoresis
Shearing can observe in contaminated DNA. The pure DNA remains intact and migrates easily into the gel so no band shearing is observed in pure DNA.
Again, it is applicable for research purposes only, not recommended for sensitive experiments such as DNA sequencing and microarray. Still, the cost of the Qubit kit will increase the per-sample cost for the experiment.
So for long-term use purchase Nanodrop light.
I personally recommended the Nanodrop light for the molecular Genetic labs because it performs both the function for qualitative analysis and quantitative analysis. Further, Nanodrop does not require any additional utilities or chemicals. It is easy to use as well. The sample processing is faster than Qubit.
Nonetheless, the Nanodrop and the Qubit both can process only a single sample at a time. In my opinion, the Nanodrop light wins the race between the comparison of Qubit vs Nanodrop.