6: Appendix III- Using the Nanodrop

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Nathan Reyna, Ruth Plymale, & Kristen Johnson
Ouachita Babtist University & University of New Hampshire

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How to Use the NanoDrop

Watch the YouTube videos regarding questions for the NanoDrop.

(https://www.youtube.com/watch?v=y3ICiycWeCA&t)

The NanoDrop allows you to measure the concentration of DNA, RNA, and protein in a couple of microliters of the solution. It is best to use 1.5\(\mu L\) to 2\(\mu L\) for accurate measurements. Some models have the ability to measure fluorescently labeled molecules.

Procedure:

Wash the NanoDrop pedestal. Lift the upper arm of the NanoDrop and remove the wipe.
Add 4‐5 \(\mu L\) of purified water to the lower pedestal, then lower the arm.
Wait 30‐60 seconds.
Lift the upper arm and use the wipe to vigorously scrub both the upper and lower pedestals.
Open the NanoDrop software on the computer by double-clicking the “ND-1000” icon that looks a bit like an hourglass
Initialize the NanoDrop:
Click on the “Nucleic Acid” button in the NanoDrop software. This brings up q dialog box; DO NOT click “okay” until you have added water.
Add 2 \(\mu L\) of purified water to the lower pedestal, then lower the upper arm.
Click “okay” on the computer and wait about 20 seconds while the NanoDrop initializes
When it is done, lift the upper arm and dry it off with a wipe
Blank the NanoDrop:
Add 2 \(\mu L\) of the buffer your sample is in (most likely elution buffer).
Lower the upper arm of the NanoDrop and click the “Blank” button on the software.
Wait about 20 seconds for the blank measurement to be made.
Lift the upper arm and dry the pedestal with a wipe
Measure your Sample:
Add 2 \(\mu L\) of your sample to the lower pedestal, then lower the upper arm.
In the “Sample ID” box, type the name of your sample.
Click the “Measure” button on the software and wait about 20 seconds for the measurement to be complete.
Lift the upper arm and dry the pedestal OR lift the upper arm and carefully pipet up as much of the sample as you can to retain for further use.
Collecting Data:
Write down any measurements you are interested in.
Click the “Print Screen” button to print the complete spectrum if desired.
When finished making all measurements, click “Print Report” to get a table of all data.
Cleaning the Pedestal:
Add 4-5 \(\mu L\) of purified water to the lower pedestal, then lower the arm.
Wait 30-60 seconds.
Lift the upper arm and use a wipe to vigorously scrub both the upper and lower pedestals.
Place a new folded lab wipe on the lower pedestal and close the upper arm.
Shut down and close the NanoDrop computer.

More information on what these NanoDrop values mean:

https://tools.thermofisher.com/content/sfs/brochures/T123-NanoDrop-Lite-Interpretation-of-Nucleic-Acid-260-280-Ratios.pdf