Established in the mid 1970's, New England Biolabs, Inc. (NEB) is the industry leader in the discovery and production of enzymes for molecular biology applications and now offers the largest selection of recombinant and native enzymes for genomic research. NEB continues to expand its product offerings into areas related to PCR, gene expression, sample preparation for next generation sequencing, synthetic biology, glycobiology, epigenetics and RNA analysis. Additionally, NEB is focused on strengthening alliances that enable new technologies to reach key market sectors, including molecular diagnostics development. New England Biolabs is a privately held company, headquartered in Ipswich, MA, and has extensive worldwide distribution through a network of exclusive distributors, agents and seven subsidiaries located in Canada, China, France, Germany, Japan, Singapore and the UK. For more information about New England Biolabs visit neb.
Quantifying a DNA, RNA or protein sample concentration is now as easy as a click of the pipette, a push of a button and a dab of tissue to clean up. Here’s what you need to know about a few of the strengths and limitations of your Nanodrop – before you set up.
Take a number, please…
A Nanodrop is a common lab spectrophotometer (you may already be familiar with the 1000 or 2000 model) that reads a single 2μl drop on a pedestal. Less prep and cleanup time means you’re able to measure several samples in under a minute, compared to what’s needed to read just one sample in a traditional cuvette! This is perfect for most lab applications, but if you’re quantifying more than 96 samples at a time for microarrays, genotyping or to send off to a core facility, you might want to utilize a Nanodrop 8000 that can read up to 8 samples simultaneously.
Tell me the source: DNA, RNA or Protein
The Nanodrop does an excellent job at measuring across a wide spectrum that spans UV and visible light. It can’t automatically determine for you that the sample on the pedestal is DNA, RNA or protein – you have to tell the software before beginning measurements so it can report an accurate concentration. Though if you happen upon a circumstance where you’ve measured half of your RNA samples at the default setting (DNA for us) you can start over again by rereading each sample at the correct software setting, or you can estimate your sample concentration by hand, based upon the raw absorbance data already collected using the table below for guidance. Remember to correct for your blank TE or water sample!
|The concentration at an A260 value of 1.0 for|
|Double stranded DNA||50 μg/ml|
|Single stranded RNA||40 μg/ml|
|Single stranded DNA||33 μg/ml|
|Protein (measured at A280)||1 mg/ml|
Please note that these are for relatively pure samples of DNA, RNA and protein at an A260/280 of about 1.8, 2.0 and 0.6, respectively. Additionally, the concentration of your sample will be reported in ng/μl.
Quick and dirty sample extractions: pump it up
Regardless of quantification method there are two important questions to ask after every nucleic acid extraction: Is there contamination in my sample tube? If there is, what kind is it?
From what may be read as a perfect DNA sample based upon the A260/280 alone, your Nanodrop may be overstating the results. This is a result common to most spectrophotometers: your equipment is doing what it was designed to do and that’s read absorbance at specific wavelengths.
But DNA is not the only item that absorbs at 260 nm. RNA does too. Phenol is close at about 270 nm and all of this combined has the potential to push your reported concentration upwards.
Become aware of contamination that you can see
Another point of interest is your A260/230 value, which like its A260/280 counterpart should land at or above 1.8 as a general rule of thumb, and above 2.0 for relatively pure DNA and RNA samples.
- Low A260/280, large peak at A280: protein contamination
- Large A230: phenol again, EDTA or carbohydrate contamination
- Small peaks or higher than expected absorbance along the far right side of the graph: contamination stemming from transfer of the interface during phase separation step in extraction.
- From the source, here’s what it can look like on your screen: 260/280 and 260/230 Ratios (ThermoScientific/Nanodrop).
One way to ensure a cleaner sample is to send it through to re-precipitation, followed by an ethanol wash, extended air-drying and re-suspension in a fresh volume of TE or pure water. Yes, your reported concentration may be significantly less than before, but that’s because you have successfully removed any contamination that was impacting your absorbance data for better or worse.
Look out below!
For the most part your nucleic acids extractions land within the measurable range of the Nanodrop (2ng – 15μg per μl). It’s when you start extracting from single cell or small sample sources that you need to consider alternative quantification methods. For example, fluorescent-dyes will help resolve amounts of DNA or RNA at or below 2ng/μl.
Want to learn more about DNA, RNA and your Nanodrop? Check out these other interesting resources:
- Nucleic Acid Quantitation (Wikipedia)
- Nanodrop (OpenWetWare)
- Quick reference: Determining DNA Concentration & Purity
- Spectrophotometer for nucleic acid measurements
- Assessment of Nucleic Acid Purity
- Where did my DNA go? Tips on improving DNA Clean-Up
- DNA Precipitation: Ethanol vs. Isopropanol
- Ethanol Precipitation of DNA and RNA: How it works