How to Calculate the Purity of Dna From Absorbance Reading
Decide the concentration and purity of nucleic acids
Written by Éva Mészáros
24. June 2021
Purifying nucleic acids from samples is a mutual workflow in molecular biology labs. We've put together a v-role serial explaining what you need to consider when setting up a lab and ownership equipment for it to help you take the right decisions. After discussing efficient sample collection, nucleic acrid extraction methods and the set-up of a PCR lab, nosotros'll at present take a expect at different methods to determine the concentration, yield and purity of nucleic acids.
Tabular array of contents
1 Agarose gel electrophoresis
Gel electrophoresis
Gel electrophoresis is a technique used to separate molecules like DNA, RNA or proteins based on their size and charge. Samples are loaded into wells at one terminate of a gel, and an electric current is applied. The molecules travel faster or slower through the gel depending on their accuse and size, assuasive them to be separated from one some other.
Gel electrophoresis can be performed with unlike types of gels, each of which is suitable for a different sample blazon. Nucleic acids are usually analyzed with an agarose gel.
1.i How does it work?
For agarose gel electrophoresis, y'all starting time bandage a gel by dissolving agarose – a natural polysaccharide derived from a type of seaweed1 – in a conductive buffer and allowing it to gear up in a gel tray. Apply a plastic comb to create sample wells. Once the gel has prepare, place the tray in a gel tank filled with a conductive buffer solution or add together the buffer solution into the gel tray, mix your nucleic acid samples with a loading dye and pipette them into the sample wells. To compare the size of your nucleic acids with a molecular-weight size marker, add together it to the first well. Then employ an electrical field along the length of the gel. Equally the backbones of the nucleic acids are negatively charged, they will migrate towards the positively charged electrode and separate depending on their size – the bigger the nucleic acids the slower they migrate. Afterward running the gel, visualize your nucleic acids by staining the gel with a fluorescent intercalating dye such equally ethidium bromide and capture an paradigm with a gel documentation system. Depending on the dye, you may need to add it before casting the gel.1
i.2 Equipment and buffer needed
To perform agarose gel electrophoresis, you demand a gel electrophoresis system, an external power supply and a biosafety cabinet every bit the intercalating dyes are chancy.
The two well-nigh mutual buffers for agarose gel electrophoresis are TAE (Tris-acetate-EDTA) and TBE (Tris-borate-EDTA). The differences betwixt the two are that TAE is better at separating large nucleic acid segments (>15000 bp) while TBE is well suited for smaller fragments (<thousand bp). TBE should besides be chosen for long or repeated runs, as it has a better buffering chapters and is therefore less decumbent to overheating. Nevertheless, as borate is an enzyme inhibitor, you lot have to use TAE if you desire to recover the bands and apply the nucleic acids for downstream applications involving an enzyme, such as PCR. And, most importantly, ensure that you don't mix the dissimilar buffers, e.g. by using TAE to bandage your gel and then placing the tray in a tank filled with TBA.2,3,4,five
ane.3 How to determine the concentration and purity
Using agarose gel electrophoresis, the nucleic acid concentration of your sample can merely exist roughly estimated by comparing the intensity of your nucleic acrid band with the corresponding band of the size marker. If yous for case load a 2 µl sample of undiluted Dna on the gel and your band has near the same intensity equally the band from the 100 ng standard of the same length, your sample has a concentration of 50 ng/µl (100 ng divided by ii µl).half-dozen
In addition to giving you a rough guess of the concentration of your samples, agarose gel electrophoresis helps you to check their purity afterward nucleic acid extraction protocols. If yous've for example extracted genomic Dna, you can run a gel to run across if your samples are contaminated with RNA, which could exist detected equally a depression molecular weight smear.7
Running a gel is as well recommended after PCR reactions, every bit they are highly sensitive and prone to contamination. Checking if the negative and positive controls provide the expected results will help you to place primary mix contaminations early on and confirm the functioning of the extraction protocol, reagents and amplification steps. Moreover, y'all will be able to see if only the sequence of interest has been amplified by verifying if you only get a single band. And if you utilize a size marking, you tin can validate that the amplified nucleic acrid segments accept the expected size.
2 Spectrophotometry
Spectrophotometry
Spectrophotometry measures the proportion of light of a certain wavelength that is absorbed by a sample. The absorbance values let both qualitative and quantitative analysis of the sample, due east.g. by determining the purity of a solution or the concentration of a certain analyte.
2.1 How does information technology work?
To measure nucleic acid concentration and purity using spectrophotometry, you tin either work with a microspectrophotometer or a spectrophotometer and microcuvettes.
If you're using a microspectrophotometer, you lot kickoff demand to make clean the upper and lower pedestals with distilled water past pipetting a droplet onto the lower pedestal, closing the pedestal arm, waiting for a brusk time, raising the arm and wiping the pedestals with a dry, lint-costless lab wipe.
Once you've cleaned the instrument, you should pipette a droplet of the buffer, that the nucleic acid is suspended or dissolved in, onto the lower pedestal. As shortly as you lower the microspectrophotometer'south arm, the liquid will course a cavalcade between the upper and lower pedestal, concord in identify by the surface tension. To perform the spectral measurements, the microspectrophotometer passes light from a xenon flashlamp from the upper pedestal through the liquid and the lower pedestal where it is detected by the integrated spectrometer.
Performing a blank measurement with the buffer allows to eliminate the influence of the absorbance of the buffer. Once yous've zeroed the microspectrophotometer, you can measure the absorbance of your samples at wavelengths of 230 nm, 260 nm and 280 nm. In order to get reproducible results, your samples need to exist homogenous and well-mixed. As well ensure that you lot continue the pedestals clean by wiping each sample from both the upper and lower pedestals before adding the next 1. Nosotros recommend to perform a blank measurement after every 10 samples. If it shows no absorbance, go on with the next batch of ten samples, and if you detect absorbance, remove residues from previous samples by cleaning the pedestals with distilled water.
Working with a spectrophotometer is very like, but instead of pipetting your blank and samples as a droplet onto the lower pedestal, you fill them into microcuvettes and insert them 1 by one into the spectrophotometer which will perform the absorbance measurements for you.
two.2 Equipment needed
Every bit just discussed, you tin can either utilise a spectrophotometer working with microcuvettes or a microspectrophotometer. Whereas a spectrophotometer is more sensitive, a microspectrophotometer can work with sample volumes as depression equally 0.v µl. Many manufacturers too offer devices that combine the ii.
2.3 How to make up one's mind the concentration and purity
Spectrophotometers and microspectrophotometers calculate the nucleic acrid concentration using the following formula:
Concentration (µg/ml) = A260 reading 10 conversion factor
The conversion gene is 50 µg/ml for dsDNA, 40 µg/ml for RNA and 33 µg/ml for ssDNA.
Notation: Don't forget that yous need to multiply the concentration with your dilution factor if you diluted your samples before making the measurements.
Once you know the concentration of your sample, you can calculate its yield every bit follows, e.one thousand. to decide if your PCR reaction generated a sufficient corporeality of nucleic acids for your downstream application:
Yield (µg) = Concentration ten total sample volume (ml)
Spectrophotometry can also be used to determine the purity of a sample. Calculate the A260/A280 ratio to detect protein contamination (and RNA contamination in the case of DNA samples) and the A260/A230 ratio to find contamination with chaotropic salts, EDTA, non-ionic detergents, proteins and phenol. Pure dsDNA has an A260/A280 ratio of ane.85-1.88 and pure RNA of effectually ii.ane. The A260/A230 ratio is ordinarily higher than the A260/A280 ratio and typically lies between ii.3-2.four for dsDNA and 2.1-two.iii for RNA.viii
Practise y'all like what you're reading? Stay tuned!
3 Fluorometry
Fluorometry
Fluorometry is a technique used to place and quantify analytes in a sample by adding fluorophores that bind to the analyte of interest to the sample, exciting them with a beam of UV low-cal and detecting and measuring the emitted wavelength. By comparing the sample fluorescence to known standards, the analyte can be quantified.
three.1 How does it work?
In contrast to spectrophotometry, fluorometry requires an assay set-upwards. This means that y'all demand to go an assay kit suitable for your sample and consisting of a fluorescent dye, buffer and standards. When preparing your samples, follow the kit manufacturer's instructions to ensure that the fluorescent dye binds to your nucleic acids and the standards. An overview over the virtually common fluorescent dyes can be plant in the table below. Note that it's important to avert introducing air bubbles during pipetting and mixing steps as they could later on on negatively influence the measurements of the fluorometer.
| Manufacturer | Dye for: | ||
|---|---|---|---|
| dsDNA | ssDNA | RNA | |
| Thermo ScientificTM | PicoGreenTM | OliGreenTM | RiboGreenTM |
| Promega | QuantiFluor® dsDNA | QuantiFluor® ssDNA | QuantiFluor® RNA |
| InvitrogenTM (QubitTM) | Qubit dsDNA assay | Qubit ssDNA assay | Qubit RNA analysis |
Before starting your measurements on the fluorometer, you demand to calibrate the instrument by reading the PCR tubes with the standards. The first standard should bespeak a concentration of 0 ng/ml and the last ane the maximum concentration of your analysis range. Later calibrating the fluorometer by creating the standard bend, you can add together PCR tubes containing your samples one afterward the other.9
3.two Equipment needed
Every bit explained in the section above, you need a fluorometer and an assay kit to perform fluorescence measurements. If yous take many samples to clarify, you lot can also utilize a microplate reader capable of measuring fluorescence with an integrated PC. The divergence in the assay set-upwardly would be that you add your samples and standards into a microplate instead of PCR tubes and and then measure out the fluorescence of the unabridged plate in i go.
three.3 How to determine the concentration
Fluorometers unremarkably calculate the nucleic acid concentration for you. If non, you can calculate information technology by comparing the fluorescence of your sample against the standard bend. As for spectrophotometry, don't forget to multiply the concentration you get with the dilution gene and calculate the yield by multiplying the concentration with the total sample volume.
The purity of your nucleic acids can't be calculated with fluorometry as information technology merely detects the fluorophores bound to the nucleic acids and therefore can't detect contaminants.
4 Spectrophotometry vs. fluorometry
Every bit we've just explained, fluorometers can't measure the purity of nucleic acrid samples, only their concentration. Another downside compared to spectrophotometry is that it's more than plush because of the expensive assay kits and more fourth dimension consuming as you lot need to mix your samples with the fluorescent dye before analysis.
Its advantages over spectrophotometry are that it'southward way more sensitive, providing better results with diluted samples, e.g. after nucleic acid extraction. The lower detection limit of the NanoDropTM spectrophotometer is for instance 0.4 ng/µl, whereas the Qubit fluorometer has a lower detection limit of 0.005 ng/µl.10,11 On top of that, fluorometers don't overestimate the nucleic acrid concentration as they don't detect the absorbance of other sample components, e.g. proteins. Some other advantage of fluorometry is that you can apply microplate readers capable of analyzing an entire microplate in one run, significantly increasing your throughput.
Afterward a PCR, neither the sensitivity nor the overestimation of the concentration are usually a trouble, as yous have a very loftier number of the nucleic acid segment of involvement (amplicon) anyway. This means that spectrophotometry is the preferred method after a PCR reaction, unless you lot need to determine the concentration very precisely, for example if your downstream awarding is Side by side Generation Sequencing (NGS). For NGS assays, y'all tin can pool up to 96 samples to clarify them in one go and these samples need to have the exact same concentration in order to evangelize the desired results, making fluorometry the ideal method.
Equally it's very critical to take the same nucleic acid concentration in all your samples when pooling them for an NGS analysis, you may even want to double check your fluorometry results, for example with the TapeStation organization from Agilent. It works like to gel electrophoresis, but is fully automated. Once you have inserted your samples, tips and the ScreenTape (a minor cassette containing electrodes, a gel matrix and a buffer suitable for your sample type), it can automatically analyze up to sixteen samples and will provide you with a gel prototype and a concentration measurement for each sample.12
5 What we use in our own lab
For our ain lab that we recently ready, we bought a gel electrophoresis system for the visualization of nucleic acids and a microspectrophotometer that we employ for an additional quality check and concentration measurements. Since spectrophotometry is usually precise enough for our downstream applications, we didn't purchase a fluorometer. If we yet need to perform an NGS assay, nosotros buy an analysis kit and perform the fluorescence measurements on a microplate reader that nosotros already own.
6 Conclusion
To check the success of your nucleic acrid extraction protocols and PCR reactions and to make up one's mind the purity and concentration of your nucleic acid samples, you usually demand to use a combination of methods. Gel electrophoresis and spectrophotometry are suitable for most samples and applications, but if you work with diluted samples or need highly precise concentration measurements for downstream applications such as NGS, a fluorometer may be required every bit well, and yous could even consider getting a TapeStation to double cheque the results.
We hope that this blog postal service answered all your questions on nucleic acrid visualization and quantification. But what if it turns out that your samples aren't pure enough? Read our weblog post on nucleic acid purification to get the answer.
Questions? Feel free to enquire!
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Source: https://www.integra-biosciences.com/global/en/blog/article/determine-concentration-and-purity-nucleic-acids
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