Checking DNA purity with the NanoDrop 2000C

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

Protocol

  1. Double-click on the NanoDrop 2000C software icon from the desktop.
  2. With the group set to CLASSIC, click on the option for NUCLEIC ACID.
  3. Allow the instrument to conduct its routine verification of wavelength.
  4. Wipe down the pedestal with a clean, damp Kimwipe.
  5. To blank the instrument, raise the arm and load 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.
  6. Wipe down the pedestal with dry Kimwipe.
  7. Type your SAMPLE ID for your first sample in the top right corner.
  8. Gently mix your sample first for accurate concentration reading.
  9. Load 2 µL of your sample onto the pedestal. Lower the arm and click MEASURE.

  10. 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). 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.

  11. Once you have finished reading all of your samples, wipe down the NanoDrop pedestal with a damp Kimwipe to clean it and return the arm of the instrument to the lowered position.
  12. To export your table, 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.