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
Single stranded RNA
Single stranded DNA
Protein (measured at A280)
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.
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:
Phenol extraction is a commonly used method for removing proteins from a DNA sample, e.g. to remove proteins from cell lysate during genomic DNA preparation. It’s commonly used, but not commonly understood. If you want to know how it works so you can show off to all of your friends… read on. The basic protocol […]
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