A260/A280 Ratio - Thermo Scientific NanoDrop Lite Plus User Manual

A260/A280 Ratio

Thermo Scientific
Some researchers may encounter a consistent A260/A280 ratio change when
switching from a standard cuvette spectrophotometer to the NanoDrop Lite Plus.
The three main causes for this are:
Change in sample acidity - Small changes in solution pH will cause the A260/A280
ratio to vary. Acidic solutions will under-represent the A260/A280 ratio by 0.2-0.3,
while a basic solution will over-represent the ratio by 0.2-0.3. When comparing the
NanoDrop Lite Plus to other spectrophotometers, it is important to ensure that the
pH of an undiluted sample measured on the NanoDrop Lite Plus is at the same pH
as the diluted sample measured on the second spectrophotometer.
Wavelength Accuracy of the Spectrophotometers - Although the absorbance of a
nucleic acid at 260 nm is generally on a plateau, the absorbance curve at 280 nm is
quite steeply sloped. A slight shift in wavelength accuracy will have a large effect on
A260/A280 ratios. For example, a + 1 nm shift in wavelength accuracy will result in a
+0.2 change in the A260/A280 ratio. Since many spectrophotometers claim a 1 nm
accuracy specification, it is possible to see as much as a 0.4 difference in the
A260/A280 ratio when measuring the same nucleic acid sample on two
spectrophotometers that are both within wavelength accuracy specification.
The difference in the A260/A280 ratio is important when comparing measurements
taken on the NanoDrop Lite Plus to measurements made on other
spectrophotometers.
Nucleotide Mix in Your Sample - The five nucleotides that comprise DNA and RNA
exhibit widely varying A260/A280 ratios. The following represent the A260/A280
ratios estimated for each nucleotide if measured independently:
Adenine: 4.50
Cytosine:1.51
Guanine: 1.15
Thymine:1.47
Uracil: 4.00
The resultant A260/A280 ratio for the nucleic acid being studied will be
approximately equal to the weighted average of the A260/A280 ratios for the four
nucleotides present. It is important to note that the generally accepted ratios of 1.8
and 2.0 for DNA and RNA are "rules of thumb." The actual ratio will depend on the
composition of the nucleic acid.
Note RNA will typically have a higher A260/A280 ratio due to the higher ratio of
Uracil compared to that of Thymine.
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NanoDrop Lite Plus User Guide
Helpful Information for Measuring Samples
NanoDrop Lite Plus User Guide 19