it does not matter super much, the absorbance ratio might not be >1.8, but you will get the concentration that you are supposed to get!!

For DNA measurement on the Nanodrop spectrophotometer, yes, you can use Molecular Biology grade water for the blank. There is no need to use the AE buffer.

Hello Amna Kr,

As such, it will not matter much since the AE buffer also contains nuclease-free water, but technically, if you want to set the blank, just mix the final elution volume (AE buffer used) with the volume of water used to dilute DNA sample. For example: If you used 100uL of AE buffer for elution and 50uL of NFW for dilution then in an empty tube mix 100uL of AE buffer and 50uL of NFW. This will actually serve as your proper or technical blank.

It is best to blank with the buffer in which the DNA is in solution. Using water is fine, but then a read on the PCR blank (with respect to water) should also be performed to ensure there is not significant differences in absorption between the water and the buffer.

It depends on what you’re measuring with the NanoDrop:

• For nucleic acids (DNA/RNA): Use nuclease-free water or the same buffer your sample is suspended in (e.g., TE buffer, Tris-HCl).

• For proteins (A280): Use the same buffer your protein is in.

• For dye-based protein assays (e.g., Bradford, BCA): Use the appropriate reagent blank (e.g., Bradford reagent without protein).

• For other spectrophotometric measurements: Use a blank that matches the solvent or buffer in your sample.

Always ensure your blank is clean and free from contaminants, as it directly affects your readings.