Checking DNA purity with the NanoDrop Spectrophotometer

The CCG currently maintains two NanoDrop devices: the newer NanoDrop Ultra and an older NanoDrop 2000C. Both function similarly, except that the Ultra is a self-contained unit with touchpad controls while the 2000C is operated by a separate computer. Full user guides are available from ThermoFisher (Ultra, 2000C).

Although NanoDrop spectrophotometers will display a concentration value, this is generally not considered an accurate measurement. Nucleic acid concentrations can be measured accurately using a fluorometer, such as the Qubit.

Protocol

Setup

  1. Power on the device.
    • Ultra: Press the vertical black button on the lower right side of the device and allow the instrument to conduct its routine verification of wavelength.
    • 2000C: Power on the control computer and double-clock the “NanoDrop 2000C” icon on the desktop.
  2. Select the appropriate assay.
    • Ultra: Select the “dsDNA” icon. On the “dsDNA Setup” menu, accept the default values or press “X” to proceed to measurement.
    • 2000C: With the group set to CLASSIC, click on the option for NUCLEIC ACID. Allow the instrument to conduct its routine verification of wavelength.
  3. Raise the device arm and wipe down the pedestal with a clean, damp Kimwipe. Gently lower the arm.
  4. To blank the instrument, raise the arm and load 1–2 µL of AE buffer (or water, or TE buffer, whatever your DNA is resuspended in) onto the pedestal. Lower the arm and click BLANK.

Data Collection

  1. Raise the device arm and wipe down the pedestal with dry Kimwipe.

    The NanoDrop Ultra has an “Auto-Measure” feature, in which the instrument takes a new reading automatically every time the arm is lowered. If Auto-Naming is on (ON by default; deactivate in the dsDNA Setup menu) it will also update the sample name (Sample 1, Sample 2, etc). Auto-Measure is enabled by default but can be easily toggled OFF during an experiment by pressing the running man icon. If Auto-Measure is ON, the running man icon will be bright blue. If Auto-Measure is OFF, the icon will be dark.

  2. Type your SAMPLE ID for your first sample in the top right corner.
  3. Gently mix your sample first for accurate concentration reading.
  4. Load 1–2 µL of your sample onto the pedestal, ensuring the small “eye” of the pedestal is covered. Lower the device arm. If using the 2000C (or Ultra with Auto-Measure OFF), click MEASURE.
    • Ultra: Experiments are saved automatically and can be accessed from the clipboard icon on the left navigation bar.
    • 2000C: You will be asked to name the NanoDrop workbook you are creating. Navigate to your Molecular folder, edit filename, and save workbook (leave as .twbk file).

  5. Your DNA concentration value will appear in the top right corner of the screen with the units in ng/µL. An ideal DNA concentration for PCR will be between 25–100 ng/µL. Ideal A260/280 and A260/230 ratios will be about 2.0.

    Example of results for a good quality nucleic acid sample:

    Nanodrop curve

    This useful guide to interpreting NanoDrop DNA/RNA curves is available from ThermoFisher.

  6. Once you have finished reading all of your samples, wipe down the NanoDrop pedestal with DI water and a Kimwipe. Return the arm of the instrument to the lowered position.
  7. To export your data:
    • Ultra: Insert a USB drive into the port on the front (USB-C) or rear (standard USB) of the instrument. Press “End Experiment” and follow the onscreen prompts.
    • 2000C: Click on the REPORTS option in the lower left corner. Highlight all of your samples that you wish to export and click EXPORT. Save as an .xml file (to open later in excel) on a flash drive. Close the software when done.

For samples with DNA concentrations >100 ng/µL, a dilution of 25 ng/µL should be made to work with for PCR.

The NanoDrop Ultra has an underglow light that will display the following colors. Note that a “fail” result is not uncommon when working with non-model or preserved samples, and that good data can still be obtained even when QC results are suboptimal.

Light condition Meaning
Off Powered off/Energy Saver Mode
Breathing white Powering on or initializing
Constant white Ready/standby
Sweeping blue Measuring
Constant blue Diagnostic - pass.
Constant amber Diagnostic - conditional pass.
Blinking amber Diagnostic - fail result
Red Concentration <20 ng/µL