TITLE: C-SOP-202: Genomic DNA Purity Measurement using a Nanodrop Spectrophotometer
AUTHORS: Mihir Kekre, Ben Pascoe

[DESCRIPTION]
A standard technique for performing purity measurements is UV absorbance with a spectrophotometer. Microvolume spectrophotometers are commonly used for the analysis of nucleic acid samples. They require a small sample volume (0.5–5.0 μl) and are economical, convenient and widely available. Typically, they measure concentration and purity readings for ssDNA, dsDNA and RNA and can provide meaningful insights into the quality of the sample.

Due to some reported limitations with this method, we would recommend deploying Nanodrop UV spectrophotometry ONLY for purity ratio estimations (A260/280 and A260/230) of double-stranded or single-stranded DNA/RNA, especially when destined for downstream applications such as DNA library preparation for whole-genome, amplicon and targeted sequencing.

As an indicator of sample purity, the ratios of the absorbance values at 260 nm vs 280 nm (A260/A280) and at 260 nm vs 230 nm (A260/A230) need to be determined for each sample to ensure its suitability for downstream applications. The A260/A280 provides insight into the type of nucleic acid present (dsDNA or RNA) as well as an indication of purity. Typically, protein contamination can be detected by a reduction in this ratio; RNA contamination can be detected by an increase in this ratio. In buffered solutions, pure dsDNA has an A260/ A280 of 1.85–1.88. 

The A260/A230 is a sensitive indicator of contaminants that absorb at 230 nm. These contaminants are significantly more numerous than those absorbing at 280 nm, and include chaotropic salts such as guanidine thiocyanate (GTC) and guanidine hydrochloride (GuHCl), EDTA, non-ionic detergents like Triton™ X-100 and Tween®  20, proteins, and phenol. Substances like polysaccharides or free floating solid particles like silica fibers absorb at this wavelength, but will have a weaker effect. In buffered solutions, pure dsDNA has a higher A260/A230 ratios at 2.3–2.4. A260/A230 ratios typically produce a higher standard deviation than A260/ A280 ratios and should be interpreted with care.

This protocol has been adapted from nucleic acid purity measurement assays developed by ThermoFisher Scientific.


This is an open-access protocol distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike.
 

[STEPS]
SECTION: Before Starting
1. An initial cleaning of measurement surfaces with nuclease-free (NF) water is recommended prior to making the blank measurement.  

To clean the pedestal, pipette 2 µL of NF water onto the pedestal and lower the arm. Leave to sit for 1 min and then wipe away with lint-free tissue. 
Fig. 1a and 1b illustrate the loading technique and how the droplet sits on the pedestal.

A final cleaning of both measurement surfaces with nuclease-free (NF) water is also recommended after the last sample measurement.
 

   

   

SECTION: Initialisation
3. Open up the Nanodrop software from the desktop shortcut. Choose the type of sample to be measured (in this case, Nucleic Acid , then dsDNA)
SECTION: Initialisation
4. At the prompt, pipette 2 µL of NF water onto the pedestal and click OK. This will complete initialisation of the Nanodrop instrument.

SECTION: Blank Measurement
5. Once again, select the type of sample that needs to be measured. Load 2 µL of the blanking buffer onto the lower measurement pedestal and lower the sampling arm.
 

 


SECTION: Blank Measurement
6. Click Blank to measure and store the reference spectrum.  

SECTION: Blank Measurement
8. Pipette another 2 µL of the blanking buffer onto the pedestal, lower the arm and click Measure. The result should be a spectrum that varies no more than 0.04 Abs (10 mm absorbance equivalent) from the baseline at 260 nm. If not, clean the measurement surfaces and repeat steps 1-5.

SECTION: Sample Measurement
9. After the blank measurement is complete, enter the sample name in the Sample ID box, and choose the appropriate sample type as indicated below in Table 1:

  

SECTION: Sample Measurement
10. Pipette 2 µL of the gDNA sample onto the lower measurement pedestal and lower the sampling arm. Click Measure.
 



SECTION: Sample Measurement
11. After the measurement is complete, use a dry, lint-free lab wipe to remove the buffer from both the top and bottom measurement surfaces (Fig. 2).
SECTION: Sample Measurement
12. Review spectral image to assess sample quality (Fig. 3).

   
Refer to the Troubleshooting section on page 17 of the Nanodrop Nucleic Acid Technical Guide for information on aberrant spectral patterns.

SECTION: Sample Measurement
13. Note down the purity ratios: A260/280 and A260/230.

Refer to the Purity Ratios section on page 14 of the Nanodrop Nucleic Acid Technical Guide for information on expected values ranges.
 

 

 

SECTION: Sample Measurement
14. Repeat steps 7, 10-12 for each of the next samples.
 

SECTION: Blank Measurement
7. After the measurement is complete, use a dry, lint-free lab wipe to remove the buffer from both the top and bottom measurement surfaces (Fig. 2).

  

SECTION: Additional Information &amp; Troubleshooting
15. Technical Bulletin (ThermoFisher): Interpretation of Nucleic Acid Ratios

Nanodrop Nucleic Acid Technical Guide

Technical Note (New England BioLabs): A practical guide to analysing nucleic acid concentration & purity using MVS

Nanodrop 2000 User Manual

Nanodrop 8000 User Manual

Nanodrop One User Guide
SECTION: Before Starting
2. Prior to initiating the protocol, ensure that all active workbenches are cleaned with 80% ethanol, all relevant personal protective clothing is worn and the work area is prepared for DNA quantification according to local GLP guidelines.


Create an organised bench space by clearing away all clutter in order to maximize work efficiency.