Spectrophotometry Lab # 2
UV Spectrophotometry of DNA, RNA, and Proteins
Purpose
Molecular biologists routinely work with DNA, RNA, and proteins and have devised
some simple, fast spectrophotometric assays for these molecules. The purpose of this
exercise is to use the UV absorbance of biological samples to obtain qualitative and
quantitative information about those samples. In this spectrophotometry exercise you
will:



Measure the absorbance at 260 nm to quantify DNA.
Calculate A260/A280 ratios to estimate the purity of a DNA preparation.
Operate a spectrophotometer in the UV range.
Brief Background Review
A. The UV Absorbance Spectra of Nucleic Acids and Proteins
Most biological molecules do not intrinsically absorb light in the visible range, but they
do absorb ultraviolet light. Biologists take advantage of UV absorbance to quickly
estimate the concentration and purity of DNA, RNA, and proteins in a sample. It is
possible to quantify the amount of DNA in a sample by looking at its absorbance at a
wavelength of 260nm or 280nm (in the UV region). The UV method described in this
exercise is not highly accurate but it is very widely used since it is easy, quick, and little
DNA is required.
Proteins have two absorbance peaks in the UV region, one between 215-230 nm, where
peptide bonds absorb, and another at about 280 nm due to light absorption by aromatic
amino acids (tyrosine, tryptophan and phenylalanine). Certain of the subunits of nucleic
acids (purines) have an absorbance maximum slightly below 260 nm while others
(pyrimidines) have a maximum slightly above 260 nm. Therefore, although it is common
to say that the absorbance peak of nucleic acids is 260 nm, in reality, the absorbance
maxima of different fragments of DNA vary somewhat depending on their subunit
composition. Figure 1 shows the UV spectra for DNA, RNA, and proteins.
Observe that although proteins have little absorbance at 260 nm, both proteins and
nucleic acids absorb light at 280 nm. Therefore, if nucleic acids and proteins are mixed in
the same sample, their spectra interfere (overlap) with one another.
Table 1 below summarizes the UV wavelengths relevant to the measurement of nucleic
acids and proteins
FIGURE 1
Figure 1. UV ABSORBANCE SPECTRUM FOR DNA AND PROTEINS. A. DNA.
b. Protein (BSA). C. The absorbance spectrum for a mixture of DNA and protein.
Distinct DNA and protein peaks cannot be resolved.
TABLE 1
UV MEASUREMENTS OF DNA, RNA AND PROTEINS
WAVELENGTH
SIGNIFICANCE
COMMENTS
215-230 nm
Minimum absorbance for Measurements are generally
nucleic acids
not performed at this
Peptide bonds in proteins wavelength because
absorb light
commonly used buffers and
solvents, such as Tris, also
absorb at these wavelengths.
260 nm
Nucleic acids have
Purines absorbance
maximum absorbance
maximum is slightly below
260; pyrimidines maximum.
is slightly above 260.
Purines have a higher molar
absorptivity than
pyrimidines. Therefore, the
absorbance maximum and
absorptivity of a segment of
DNA depends on its base
composition.
Proteins have little
absorbance at this
wavelength.
270 nm
Phenol absorbs strongly
Phenol may be a
contaminant in nucleic acid
preparations.
280 nm
Aromatic amino acids
Nucleic acids also have
absorb light
some absorbance at this
wavelength.
320 nm
Neither proteins nor
Used for background
nucleic acids absorb
correction since neither
nucleic acids or proteins
absorb at this wavelength.
B. Concentration Measurements of Nucleic Acids and Proteins in a Sample
Determining concentration in a “pure” sample containing only proteins or nucleic acids,
involves constructing a standard curve.
It is possible to determine the concentration of nucleic acids or proteins based on their
absorbance at a wavelength of 260 nm or 280 nm respectively. A calibration curve using
standards of known concentration can be constructed. For accurate results, the standard
curve should be prepared using the protein of interest or DNA that is similar to that in the
sample being measured. The linear range for DNA values is reported to be from about 550 µg/mL. Depending on the protein, UV analysis of proteins at 280 nm has a linear
range from about 0.1 - 5 mg/mL.
The average absorptivity constants for proteins and nucleic acids lead to the following
relationships:




If a sample containing pure double-stranded DNA has an absorbance of 1 at
260 nm, then it contains approximately 50 µg/mL of double -stranded DNA.
If a sample containing pure single-stranded DNA has an absorbance of 1 at
260 nm, then it contains approximately 33 µg/mL of DNA.
If a sample containing pure RNA has an absorbance of 1 at 260 nm, then it
contains approximately 40 µg/mL of RNA.
Values for proteins vary. A very rough rule is that if a sample containing
pure protein has an absorbance of 1 at 280 nm, then it contains
approximately 1 mg/mL of protein.
For example, 1 mg/mL of bovine serum albumin is reported to have an A280 value of 0.7.
Antibodies (which are a type of protein) at a concentration of 1 mg/mL are reported to
have an A280 between 1.35 and 1.2. (Values from "Antibodies: A Laboratory Manual", by
E. Harlow, and D. Lane, p. 673. Academic Press, New York., 1988.)
C. Estimation of the Purity of a Nucleic Acid Preparation
It is possible to use UV spectrophotometry to estimate the purity of a solution of nucleic
acids. This method involves measuring the absorbance of the solution at two
wavelengths, usually 260 nm and 280 nm, and calculating the ratio of the two
absorbances:



An A260/A280 ratio of 2.0 is characteristic of pure RNA.
An A260/A280 of 1.8 is characteristic of pure DNA.
A260/A280 ratio of about 0.6 is characteristic of pure protein
Therefore, a ratio of 1.8 - 2.0 is desired when purifying nucleic acids. (Note that this
method does not actually distinguish DNA and RNA from one another.) A ratio less than
1.7 means there is probably a contaminant in the solution, typically either protein or
phenol.
Table 2 below summarizes the various UV methods described in this exercise.
TABLE 2
APPROXIMATING THE CONCENTRATION AND PURITY OF DNA, RNA OR
PROTEIN IN A SAMPLE
 Concentration of double-stranded DNA ~ (50 µg/mL) X (absorbance at 260
nm)
 Concentration of single-stranded DNA ~ (33 µg/mL) X (absorbance at 260
nm)
 Concentration of RNA ~ (40 µg/mL) X (absorbance at 260 nm)
Turbidity causes an apparent increase in the absorbance of a sample leading to
incorrect readings. To compensate for slight turbidity, a background correction
can be used. Proteins and nucleic acids do not absorb at 320 nm. Therefore, if a
sample absorbs at 320 nm, the absorbance is due to turbidity. The absorbance at
320 can be subtracted from the readings at 260 nm and 280 nm:
 Concentration of double-stranded DNA ~ 50 µg/mL (A260 - A320)
 Concentration of single-stranded DNA ~ 33 µg/mL (A260 - A320)
 Concentration of RNA ~ 40 µg/mL (A260 - A320)
 Concentration of protein ~ 1 mg/mL X the absorbance at 280 nm
 Concentration of protein ~ 15 mg/mL X the absorbance at 215 nm
Notes: Values for proteins vary. The rule that 1 mg/mL of protein has an
absorbance of 1 is approximate.
Tris and other common solvents also absorb light at 215 nm. For this reason, 280
nm is far more commonly used for protein measurements.
 Purity
An A260/A280 ratio of 2.0 is characteristic of pure RNA
An A260/A280 of 1.8 is characteristic of pure DNA
An A260/A280 of 0.6 is characteristic of pure protein
Notes: A ratio of 1.8 - 2.0 is desired when purifying nucleic acids.
This method does not actually distinguish DNA and RNA from one another.
A ratio of less than 1.7 means there is probably a contaminant in the solution, usually
either protein or phenol.
Laboratory Activities
Use quartz cuvettes in the UV range. Handle them with caution!
1. Measure the A260 and the A280 for each sample. Calculate their A260/A280 ratios.
2. Calculate the concentration of the DNA, RNA, and protein samples based on their
A260 values. Record the concentrations of these samples based on what is written
on their labels.
3. Place results for the class on the board. Include:
a.
A260 and the A280 for each sample.
b.
A260/A280 ratios for the samples.
c.
Your calculation of the concentrations of DNA, RNA, and protein in the samples
based on the A260 values.
4. If your results differ from those of your classmates, try to figure out why. If all students use
the same samples, the results should be the same.
5. Try mixing some protein with one of the DNA or RNA samples to see what this does to the
absorbance values.
SUMMARY OF RESULTS
UV SPECTROPHOTOMETRY OF DNA, RNA AND PROTEINS
DNA
A260
A280
A260/A280
Purity?
Concentration
RNA
A260
A280
A260/A280
Purity?
Concentration
PROTEIN
A260
A280
A260/A280
Purity?
Concentration
MIXTURE
A260
A280
A260/A280
Purity?
Concentration
Discussion:
1. What is the estimated concentration of DNA, RNA and protein in your samples based on their
absorbances at 260 nm? The DNA, RNA, and protein solutions should be labeled with their
concentration based on how much the preparer weighed out and dissolved. Generally we make
these at 0.1 mg/mL for the DNA and RNA, and 1 mg/mL for the protein. Compare the estimated
concentrations based on absorbance to the expected concentration based on the preparation of the
samples. Are they the same? If not, assume that the concentrations labeled on the solutions are the
correct concentrations.
2. These UV assays are very commonly used by molecular biologists. Why? What information
is obtained from these simple assays?
3. What are the advantages and disadvantages of these UV methods? Consider their accuracy,
precision, range, ease of use, cost, and any other factors you think are important.
Group
DNA
A260, A280
A260/A280
DNA
[]
based on
A260
RNA
A260, A280
A260/A280
RNA
Protein
[]
A260, A280
based on A260 A260/A280
Mix
A260, A280
A260/A280
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