Personally, I believe the Nanodrop is one of the most important, underrated pieces of equipment in the lab. By using just 1 μL of your sample, this nifty little machine will tell you the concentration and purity of your nucleic acids. All of this without paying for additional reagents!
Here are some tips when using the Nanodrop to measure nucleic acids in your sample:
1. Always clean the pedestal before use
In our lab, it is best practice to clean the Nanodrop’s pedestal before and after you use it. However, be aware that not everyone is on the same wavelength, excuse the pun. Always clean the pedestals before turning the Nanodrop on by pipetting 2 μL of distilled water onto the lower pedestal and closing the upper arm. Leave for this for at least 1 minute before wiping both pedestals with a lint-free tissue.
2. Always measure your blank
After blanking the Nanodrop, I find it useful to then measure the blank to see if the Nanodrop is indeed clean. If you see any peaks in your recordings you should clean the Nanodrop once again and re-blank the machine. You need to ensure your blank is exactly that, a sample free from nucleic acids and contaminants. If not, your sample values will suffer.
3. Check for air bubbles
Since the Nanodrop can use volumes as low as 1 μL to measure the absorbance of the sample, any tiny air bubbles present in the sample will wreak havoc on your results. Take care when pipetting and ensure all of the sample is deposited onto the centre (the black dot) of the bottom pedestal. Also, place the pedestal arm down on the sample slowly. Taking a little extra time to ensure your sample is bubble-free is good practice.
4. Mix your samples before measuring
This is an important step, particularly when measuring samples that have just come out of the freezer. Nevertheless, you should apply mixing to all of your samples regardless of their prior state. Give the tubes a brief vortex when the sample is completely thawed to re-homogenise the sample and pulse spin in a minifuge to pull everything to the bottom. This will ensure your readings are reproducible.
5. Be wary of low concentration samples
Despite the Nanodrop being an absolute godsend in the lab, it does have a small number of limitations, especially regarding sensitivity. The reported limit of detection for a Nanodrop 2000 is 2 ng/μL, but I would be careful when analysing samples below 10 ng/μL. Anything lower will require a more sensitive technique, such as qPCR or the Qubit. Or maybe repeat the DNA/RNA extraction and elute in a lower volume to increase the concentration of the sample.
6. Re-blank the machine every 30 minutes
When measuring many samples, it can take a good hour to get through them all considering you can only measure one sample at a time. The Nanodrop should prompt you to re-blank the machine 30 minutes after the original blank. This is to maintain consistency relative to the blank for all your samples. So, listen to the machine, and re-blank before proceeding.
7. Take measurements in duplicate at least
Since the Nanodrop only uses a single microlitre of sample, there is no excuse to not repeat measurements in duplicate. If your replicates are completely off, try cleaning the machine and check for air bubbles. Also, make sure the sample is homogenous by mixing.
8. Always write down your results
Despite most Nanodrops now supporting USB drives to export your results, I always find it best to write them down as I go along. The main results to take note are: the concentration, 260/280 ratio and 260/230 ratio. By writing them down, this keeps a paper copy of your results incase the machine breaks, as you never know what may happen.
By using these 8 simple tips when using the Nanodrop to analyse your DNA/RNA samples, you will improve the consistency and reliability of your results.
Have you got any tips you can share and add to the list? Leave a comment below.
Featured image credit: Fabian Bromann (via Flickr)
Hi there Steve, thanks for this article. Been following up on this page since I started extracting total RNA for my project. I’ve got one question. I checked the concentration (Nanodrop) of the extracted RNA samples and got concentrations of -2.8ng/ul to -2.3ng/uL. As for the 260/280, it ranges from 1.40-1.73. For 260/230, it ranges from 1.06-1.26.
Wanted to ask your opinion on this, what could possibly be the problem that gives me negative values on my concentrations.
Cheers,
Darvin
Hi Darvin,
So negative concentrations can be due to concentrations of RNA that are very low. The sensitivity of a Nanodrop is around 10 ng/uL, so anything below this will cause strange results. Alternatively, have you tried to clean (with water) the Nanodrop and re-blank the machine? This has also helped to correct strange results.
If your sample contains a low concentration of RNA then try using a more sensitive technique to quantify the sample, such as the Bioanalyzer.
I hope that helps.
Steven
Hi Steven, thanks for the helpful article. I also read your other article about interpreting the nanodrop data. Is there any advice for improving my 260/230 purity? I always get very low 260/230 (about 1.2 and sometimes it even goes below 1.0). I tried waiting a lot more than usual before dissolving my pellet into DEPC water to completely get rid of the ethanol, but the result was the same. I’m not getting the best PCR results right now and think the reason is the low purity of my cDNA.
If you could give me any advice, that would be so nice. Thanks again for the nice article
Hi Emily,
How do you do your RNA extractions? Is this with the TRIzol method? If so, you can add more ethanol washes (2 in total). This should help clean your pellet to remove any unwanted salts.
Many thanks,
Steven
Hi Steven I’ve been reading your guide for RNA extraction and this and I have a dumb question, how so deep I must to pipette since the surface sample, I mean, I must to take still the bottom or surface, maybe 3 or 4 mm? Obviously, before vortex and spin down, strictly for reproductible results.
Thanks a lot
Hi Victor
Many thanks for your message.
I would pipette from the middle of the solution if you can.
I hope that helps.
Steven
Hello,
I’ve used ddH2o water instead of the EB as a blank. would this significantly change my ratios!?
Thank you
Hi Jennifer,
I have done this before myself 🙂 Ideally you need to use the same liquid which you resuspended your sample in, but no, your ratios shouldn’t be significantly different between ddH20 and EB. Maybe run a sample resuspended in each and see how they compare.
Thanks,
Steven