Nanodrop/Qubit
‘a lot of money and effort for a result you’ll never trust’
Image Source: https://www.labtech.com/thermofisher/nd2000/
What are they and why do we use them? What types are there?
Nanodrop/Qubit are the two dominant brands of UV-visualisation spectrophotometers that most biotech labs use for research. They use UV light to measure the optical density of liquids. This can allow a researcher to gain a quantitative value for DNA, RNA and Protein concentration.
HOWEVER. While it can be nice to quantify the amount of DNA sample in a prep, the numbers provided by these devices are very rarely perfect. All of these devices are extremely susceptible to minor changes in sample prep and operation, and cross-brand comparison of results is difficult due to minor proprietary differences.
While there are now generic options available, the results obtained from a new device can only be internally compared to results from the same device. Comparisons to the results of a lab with a different device won’t be particularly useful.
When do you use?
When attempting to quantify the ratios of any of DNA:RNA:Proteins prior to downstream experiments. These results can be used to balance the quantity of sample added to each well of a gel.
When using the Nanodrop to quantify DNA: I like to use the nanodrop to quickly determine how much sample to load on an agarose gel. I then use the relative brightness of the bands on the agarose gel to tell me whether I can trust the machine. Evidence of contamination by proteins or RNA on the agarose gel will also let me know whether the result may be affected
How do you use?
Ensure the machine has been cleaned and calibrated. You will need Kimwipes, a < 10 µl micropipette and sterile tips, RO water (or the buffer your sample is stored in) and your samples.
Turn on the machine and software. Select the type of sample you wish to measure (DNA, RNA or Protein) in the software.
Zero the machine using water or the appropriate buffer. This may require several tries.
Lift the arm
Clean the nozzle, top and bottom, using a Kimwipe.
Add a 1 µl droplet to the tiny hole between the arm and the platform.
Close the arm.
Press ‘Zero’ on the software.
It may ask you to Rezero.
Lift the arm, clean the nozzle with a Kimwipe, add a 1 µl droplet to the tiny hole and close the arm.
Note the absorbance and the ratios. The relevant ratios will change depending on what you are measuring and will let you know how trustworthy the device is.
E.g. After DNA/RNA purification;
A 260/280 ratio of ~1.8 is generally accepted as “pure” for DNA
A 260/280 ratio of ~2.0 is generally accepted as “pure” for RNA. Ratios that differ from these expected values indicate contamination by phenol (e.g. ethanol), guanidine salt, or one of the other reagents used during purification.