1. Discuss the A260/A230 ratio as another key measure of relative purity.

 The 260/230 ratio is widely used as a secondary measure of DNA purity. 6,7 Expected 260/230 values for “pure” DNA are
commonly within the range between 2.0 and 2.2. If the ratio is appreciably lower than expected, it may indicate the
presence of contaminants that absorb at 230 nm such as proteins, 8 guanidine HCL (used for DNA isolations), EDTA,
carbohydrates, lipids, salts, or phenol. 9 The 260/230 ratio is considered a questionable DNA quality indicator because of
the instability of this value when a saline elution buffer is used to dissolve the DNA. It is due to the higher increase of salt
concentration than DNA concentration in the sample. Consequently, out of two DNA samples with the same purity, the
less concentrated sample will show lower 260/230 ratio because of salts absorbance at 230 nm.

It has been reported that DNA absorption depends on the solvent used. Acidic solutions will under represent the
260/280 ratio by 0.2–0.3, whereas a basic solution will over represent the ratio by 0.2–0.3. Therefore, if comparing the
260/280 ratio for different DNA samples, it is important to ensure that the pH and ionic strength of the elution buffers
used are the same.10 Moreover, absorbance at 260 nm and the 260/280 values are reproducible when low-salt buffer is
used as the elution buffer, but not water.

Aguilar, G., et. al. (2016). DNA Source Selection for Downstream Applications Based on DNA Quality Indicators Analysis.

Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4991598/

A260/A280 < 1.8 indicates polypeptide contamination and A260/A230 < 1.8 indicates presence of phenol,
polysaccharides, aromatic compounds, or polypeptides, all of which were present during extraction.13 Ethanol is a
component of the RNA wash buffer added in the final step prior to elution. A low A260/A230 ratio may be a result of not
centrifuging the spin-column in an empty dry tube for 2-3 minutes before eluting, which removes ethanol and other
leftover contaminants. Pure RNA has an A260/A230 ratio > 1.8 and an A260/A280 ratio between 1.8-2.2.

The University of Akron (2020). Assessing pepper pungency b Assessing pepper pungency by using R y using RT-qPCR t
-qPCR to measur o measure Pun1 expression. Retrieved from https://ideaexchange.uakron.edu/cgi/viewcontent.cgi?
article=2394&context=honors_research_projects

Absorbance at 260 nm (A260) gives a specific measurement of nucleic acid concentration, and the absorbance at 280 nm
(A280) and 230 nm (A230) measures protein and background absorption, respectively, as an indication of possible
contaminants. It is important to keep in mind that the 260 nm wavelength detects both RNA and DNA, and the presence of
genomic DNA contamination could lead to an over-estimation of RNA concentration. An A260 reading of 1.0 equals 40
μg·mL−1 of RNA or 50 μg·mL−1 of double stranded DNA. In general, an A260/A280 ratio of 1.8 to 2.1 at pH 7.5 indicates very
pure RNA, and a ratio greater than 1.8 is considered an acceptable indicator of good quality RNA. Pure RNA should also
give an A260/A230 ratio of 2 or slightly above; however, there is no acceptable lower limit of this ratio, as it is not clear which
contaminants contribute to a low A260/A230 ratio. Moreover, previous research and our data have both shown that there
was no significant correlation between A260/A230 and qPCR amplification efficiency.

Kuang, J., et. al. (2018). An overview of technical considerations when using quantitative real-time PCR analysis of gene
expression in human exercise research. Retrieved from https://journals.plos.org/plosone/article?
id=10.1371/journal.pone.0196438
 2. What are the advantages of using UV spectrophotometer in measuring the concentration and purity of nucleic
acids?

Spectrophotometric analysis Description: Involves measurement of absorbance at 260 nm (corresponds to

concentration of phenylalanine) and 280 nm (corresponds to concentrations of tryptophan and tyrosine) in comparison
to some protein standard (i.e., bovine serum albumin [BSA]). Background correction (A280-A320) for particle scattering
in solution can be employed. Values for A320 should be less than 0.02; otherwise presence of significant amounts of
impurities in the sample is indicated. Protein mass required for this test: 0.05 to 2 mg.

Advantages:

- Quick.
- Nondestructive.
- Less sensitive to protein structure variations than other methods.

Beerman, D. H. & Cheng, O. H. (nd) Nucleic Acid and Protein Concentration and Content: How to Measure & Why.
Retrieved from https://meatscience.org/docs/default-source/publications-resources/rmc/1997/nucleic-acid-and-
protein-concentration-and-content---how-to-measure-amp-why.pdf?sfvrsn=6540bbb3_2