You have just purified your plasmid and are ready to move on to your downstream application — but wait! Do you know how much plasmid DNA you have or how pure your sample really is?
To assess the concentration and purity of nucleic acids, such as plasmids, the go-to method is to use spectrophotometry. Microvolume spectrophotometers make it quick and easy to measure sample concentration and purity using only a tiny bit of the sample itself — so easy that this simple procedure is often taken for granted. In this post, we take a moment to review what exactly you are measuring and how to interpret your results.
Quick note before jumping in — in the world of microvolume spectrophotometers, the Thermo Scientific NanoDrop is one of the most popular options, so most of this post is based on reading results from a NanoDrop. The principles will apply to similar instruments from other companies, like the Implen NP80 or DeNovix DS-11, but some details might vary.
How it works — briefly
Spectrophotometers measure how light travels through a sample — in this case, the absorbance of light at three key wavelengths: 230 nm, 260 nm, and 280 nm. Nucleic acids exhibit a characteristic absorption spectra across this range of wavelengths with maximal absorption at 260 nm (Figure 1A). Thanks to the Beer–Lambert law, the instrument can calculate a sample’s nucleic acid concentration based on the sample’s absorption at 260 nm (A260).
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Figure 1: Characteristic absorption spectra for pure DNA and likely contaminants found in DNA samples. The blue lines in B-D represent the spectra for the specified contaminant, while the grey lines in B-D represent the pure DNA spectra for comparison. Each of these contaminants will have a slightly different impact on the spectra of a mixed sample. |
But, other molecules that may be present in the sample also absorb light in this range of wavelengths (Figure 1B-D). These other molecules can alter the overall 260 nm reading, distorting the final concentration calculation and potentially impacting downstream experiments. The usual suspects, such as proteins, phenol, or guanidine salts, exhibit their own characteristic absorbance spectra, which you can detect by examining the A260/280 and A260/230 ratios.
In common practice, a pure sample of double-stranded DNA should give an A260/280 ratio of ~1.8 and an A260/230 of ~2.0. However, let’s break down how different contaminants impact these ratios.
Protein contaminants
Whether you are purifying plasmid DNA from bacteria or cleaning up a PCR product, a common material you are trying to remove from your sample is protein. Proteins absorb light to varying degrees across the UV range used in NanoDrop systems. Peak absorption occurs below 230 nm, with a distinct secondary peak around 280 nm thanks to aromatic amino acids like tryptophan and tyrosine (Figure 1B). The A260/230 ratio can be a good indicator of protein contamination, but a number of other potential contaminants alter this ratio as well (Koetsier and Cantor, 2019). The 280 nm peak is more specific to proteins and why researchers traditionally use the A260/280 ratio as an indicator of protein contamination.
However, this ratio can be misleading. Some reports suggest that the A260/280 ratio does not drop significantly until protein levels exceed 70% of the sample content (Loughrey and Matlock, 2016). So, even samples with “good” A260/280 ratios could still have quite a bit of protein present.
Purification reagent contaminants
The reagents used during nucleic acid purification can leave behind a variety of potential contaminants, some of which are easier to detect by spectrophotometry than others. The ones most likely to impact absorbance readings are phenol, guanidine salts, and detergents (Figure 1C-D; Koetsier and Cantor, 2019). Guanidine salts and detergents have absorbance maxima around 230 nm and subsequently lower the A260/230 ratio of a sample. Ethanol carry-over from purification protocols is also a common concern, and while ethanol can reduce the A260/230 ratio of a DNA sample, its absorbance strength is low compared to other reagents, making it more difficult to detect. A particularly strong absorber at 230 nm is guanidine thiocyanate (used in many plasmid purification kits), which can dramatically reduce the A260/230 ratio at concentrations as low as 10 µM (Koetsier and Cantor, 2019). As such, a sample with a good A260/280 but a low A260/230 likely has some residual guanidine thiocyanate.
Phenol is a unique case, as pure phenol absorbs strongly at ~270 nm and exhibits A260/280 and A260/230 values that are close to those of pure DNA (Figure 1D). This property means that even samples with quite high levels of phenol (up to 600 ppm according to Loughrey and Matlock, 2017) will yield “good” A260/280 and A260/230 ratios. Instead, phenol is more likely to shift the peak of your sample's absorption spectra from 260 nm towards 270 nm. If your purification protocol uses phenol, then it is important to confirm that your sample’s absorption peak occurs near 260 nm as expected — if the peak is closer to 270 nm, you may have phenol in your sample.
RNA contaminants
DNA samples can also be contaminated with RNA (and vice-versa). Both RNA and DNA absorb maximally at 260 nm, so distinguishing between the two can be difficult by spectrophotometry.
RNA tends to yield a higher A260/280 and a lower A260/230 than DNA, so shifts in these values could hint at the presence of RNA contamination (Koetsier and Cantor, 2019). To confirm RNA in your DNA sample, you will need to use a complementary assay, such as a fluorescence based assay or running an agarose gel. In the case of plasmids, the use of RNase treatment to degrade bacterial RNAs in most purification protocols reduces the risk of RNA contamination — just be sure to check that your RNase has not expired (or been forgotten).
Improving read accuracy
In many cases, small amounts of contaminants in your sample will have minimal impact on downstream applications, but they can impair your ability to get an accurate measure of DNA concentration. This is especially problematic in low-concentration samples (samples with concentrations < 10 ng/μL), where absorbance readings are inherently more variable. If you do need to work with dilute samples:
- Take multiple readings to average out noisy measurements.
- Be cautious when interpreting purity ratios, as they can vary significantly at low concentrations.
The elution buffer you use will also affect how accurate your measurements are. While water is commonly used for elution, it can give more variable absorbance readings. A Tris-buffered solution tends to produce more consistent readings — just be sure it is compatible with your downstream applications and that you use the same solution for blanking your machine!
Finally, many NanoDrop systems now provide full-spectrum analysis, rather than just the absorbances at 230, 260, and 280 nm. Full-spectrum analysis allows the systems to better identify contaminants and to calculate corrected concentrations. However, such systems are no substitute for sound techniques, routine machine maintenance, and critical interpretation of the results.
If you see evidence of contamination, you can try re-purifying the sample you have, but if time and resources allow, you might be better off starting from the beginning. Before trying again, revisit your purification protocol. Are any of your materials expired? Did you pipette carefully? Remember to discard flow-through solutions and use clean tubes? How about spin times, were these long enough?
There are, of course, more considerations and other approaches for measuring DNA quantity and quality than we have covered here! Be sure to review resources on the specific instrument you use and check out some of the references we have provided below. And if you do have trouble with contamination, feel free to check out some of the plasmid purification resources on our blog and website.
References and Resources
References
Ahlfen SV, Schlumpberger M. Effects of low A260/A230 ratios in RNA preparations on downstream applications. Qiagen Gene Expression Newsletter. Issue 15/10. March 2010. https://www.qiagen.com/de/~/media/1ea8aec3bfa24543a28fcaea25986514.ashx.
Koetsier G and Cantor E. A Practical Guide to Analyzing Nucleic Acid Concentration and Purity with Microvolume Spectrophotometers. NEB Tech Note (2019).
Loughrey S and Matlock B. Detection of Protein in Nucleic Acid Samples Using the NanoDrop One Spectrophotometer. Thermo Fisher Scientific Tech Note (2016).
Loughrey S and Matlock B. Detection of phenol in nucleic acid samples using the NanoDrop One spectrophotometer. Thermo Fisher Scientific Tech Note (2017).
Extinction Coefficients. Thermo Fisher Scientific Tech Tip (2013).
Additional resources on the Addgene blog
- Plasmid Preps: Different Purity, Different Quantities, Different Uses
- What's the Best Way to Elute and Store Your Plasmid DNA?
- Selecting Your Plasmid Purification Kit
Resources on Addgene.org
Topics: Plasmids 101, Molecular Biology Protocols and Tips, Plasmids
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