Interpreting NanoDrop Results

Interpreting NanoDrop Results

Concentration:

Typically, we want our DNA to have a concentration of at least 500 ng/uL, or 0.500 ug/uL. The NanoDrop instrument generally gives measurements as ug/uL. Be aware of the units because sometimes our protocol requires concentrations in ng, not ug. Very dilute concentrations, less than 0.020 ug/uL, will be unreliable since it is near the limit of detection for the NanoDrop and most of the signal at that point is probably from background noise in the sample. The NanoDrop instrument measures concentration based on the absorbance at 260 nm, which is the peak absorbance for nucleic acids. This includes both DNA and RNA, however we normally put RNAse into our DNA samples to get rid of RNA, so the signal from RNA should be negligible. However, if you are having difficulties with your DNA product in later applications even though the purity ratios are acceptable, one possible explanation could be a significant amount of RNA in the sample. Even if it has been chopped up by the RNase, it is still present. Therefore, a cleanup column is suggested by Dr. Kim-Han.

Purity Ratios:

Besides just the amount of DNA in a sample, the NanoDrop also measures the amount of other possible contaminants and compares it to the amount of DNA in the sample to give you an idea of the purity of your sample. There are two different ratios that tell you about the quality of your DNA.

260/280: This compares the amount of DNA to the amount of proteins, which absorb at 280 nm. The presence of proteins indicates that your sample is not very pure, and will probably need to be cleaned up more. For DNA samples, a ratio of 1.8 is indicative of pure DNA, anything less than that has too much protein. Low 260/280 ratios can be caused by leftover phenol, lots of protein, or an extremely low concentration of DNA. High 260/280 purity ratios are not indicative of an issue, this just means you have a lot more DNA than protein, which is actually ideal.

260/230: This compares the amount of DNA to the amount of other contaminants, like phenol or ethanol, that absorb around 230 nm. This most likely means that there is still a lot of ethanol and phenol chloroform in the solution, and it wasn’t completely dry before being resuspended. Typically, these ratios are in the 2.0-2.2 range. Low 260/230 ratios can be caused by residual phenol from nucleic acid extraction or residual guanidine (often used in column based kits). High 260/230 ratios may be caused by an inappropriate blank measurement or a dirty pedestal. Make sure to clean the upper and lower pedestals with a Kimwipe wetted with distilled water, then wipe with a dry Kimwipe. Also make sure the blank you are using is similar to the solvent in which the DNA is suspended. In this lab, we almost always suspend our DNA in MBG water, so that is what you should use as your blank.

Although purity ratios and spectral profiles are important indicators of sample quality, the best indicator of DNA or RNA quality is functionality in the downstream application of interest.