09 UV Absorption

Experiment 2
Determination of DNA
Concentration and Purity by
Ultraviolet Spectrophotometry
Typically, the first thing one wants to know about a DNA preparation is its concentration and purity. Both
can be determined by measuring the absorption of ultraviolet light. DNA absorbs UV more or less strongly
depending upon the wavelength. We will be using a NanoDrop spectrophotometer, which takes
measurements at wavelengths of 260 and 280 nm, and in addition determines an absorption spectrum from
220 – 350 nm. The graph below shows that maximum absorbance occurs over a broad peak around 260 nm.
At 280nm DNA only absorbs about half as much UV.
Although 260 nm is considered to be the de facto peak, the actual peak absorbance varies somewhat from
DNA to DNA. UV absorption is a property of the bases, and each base absorbs differently. The actual peak
absorbance of a particular DNA, then, depends on its base composition. Since different DNA’s have
somewhat different base compositions, the actual absorbances will also be somewhat different.
45
Experiment 2
DNA concentration can be determined by the equation:
1 OD260 unit = 50 µg/ml
The NanoDrop automatically calculates concentrations and records them as ng/µl (1 ng/µl =1 µg/ml).
Concentrations of single-stranded DNA and RNA can also be determined by measuring absorption at 260 nm:
Nucleic Acid
ds DNA
ss DNA
ss RNA
Concentration (µg/ml) per A260 Unit
50
33
40
It is clear from the above table that single-stranded DNA absorbs more UV than double stranded DNA. This
is due to interactions between the stacked bases in double-stranded DNA. The difference can be also
demonstrated directly by comparing the OD’s of double-strand DNA and DNA that has been denatured by
boiling. The change in OD is referred to as the hyperchromic shift. Single nucleotides absorb UV more
strongly than single-stranded DNA.
Protein also absorbs UV and can be quantitated by spectrophotometry. However, as can be seen in the graph
above, there are two peaks of absorbance, one at 230 nm and the other at 280 nm. The peak at 230 nm is due
to absorbance by the peptide bonds while the 280 nm peak is due to absorbance by the rings of aromatic
amino acids (tryptophan, tyrosine, and phenylalanine). There is a significant dip in absorption at 260 nm,
where DNA absorbs maximally. 280 nm is typically used to determine the concentration of protein. In the
same way that different DNA’s have different base ratios, different proteins have different proportions of
aromatic amino acids, and the amount of absorption at 280 nm will vary from protein to protein. However, as
a rule of thumb:
1 OD280 unit = 1 mg/ml protein
In most DNA preparations, the final step is the separation of DNA from protein. Carryover protein during a
DNA prep could lead to problems with subsequent operations, such as cutting with a restriction endonuclease.
Assessment of purity is therefore important. The most commonly used assay is the A260/A280 ratio. As noted
above, DNA absorbs almost twice as much UV at 260 nm as it does at 280 nm. If there is no protein present,
the target ratio is 1.8. However, because 280 nm is a peak for protein absorption, protein contamination will
increase the A280 reading but have little effect on the A260 reading. Thus the A260/A280 ratio will be lower than
1.8.
Purity of
DNA
RNA
Protein
Target A260/A280 Ratio
1.8
2.0
0.6
Occasionally one recovers A260/A280 ratio greater than 2. The meaning of this is not easy to find in the
literature, but it appears to be related to DNA degradation and measurement of free nucleotides. Because
protein also absorbs at 230 nm, theoretically the A260/A230 ratio could be used to detect protein contamination.
However, this is rarely used because common buffers such as Tris also absorb at 230 nm. Nevertheless, the
46
UV Spectrophotometry of DNA
NanoDrop does a A260/A230 calculation. However, this measurement is adjustable and can be set for whatever
additional wavelength one desires. Some DNA extraction protocols require the use of phenol. While protein
contamination is not necessarily a fatal problem, phenol contamination most definitely is. Phenol absorbs
maximally at 270 nm and can have an impact on the A260/A280 ratio.
When assessing DNA purity it is important to understand that while the A260/A280 ratio is easy to determine and
is the most widely used method, it is not particularly robust. DNA absorbs so strongly at 260 nm that it takes
significant protein contamination to have a noticeable effect on the A260/A280 ratio. On the other hand, the
A260/A280 ratio is a particularly robust method for assessing DNA contamination of protein preparations.
Because DNA and RNA are so similar, spectrophotometry cannot be used to detect contamination of DNA by
RNA, and vice versa.
In this exercise, you will determine the concentration and purity of the E. coli that you prepared in the
previous lab. You will also compare the difference between double-stranded and single-strand DNA, and
study the effect of protein contamination on A260/A280 ratios.
Concentration and Purity
of E. coli Chromosomal
DNA
Hyperchromic Shift
Affect of Protein
Contamination on UV
Absorption Spectrum and
A260/A280 ratios
1
Use the NanoDrop to measure the optical density of the
chromosomal DNA you isolated in the previous lab.
2
Calculate the concentration and purity, and print out a graph of
the absorption spectrum.
1
Transfer 10 µl of your chromosomal DNA to a microfuge and
boil it for 30 minutes.
2
Calculate the concentration and purity, and print out a graph of
the absorption spectrum.
1
Obtain a tube of bovine serum albumin (BSA) of unknown
concentration.
2
Serially dilute the BSA to 100, 10-1, 10-2, and 10-3.
3
Make two measurements of OD at 280 nm, once using the Nucleic
Acid program on the NanoDrop, and once using the Protein
program.
4
Mix each of the diluted BSA samples with an equal amount of
undiluted DNA.
5
Measure the concentrations and A260/A280 ratios of the mixed
samples using the Nucleic Acid program on the NanoDrop, and
print out the absorption spectrum for each.
47
Experiment 2
Analysis
1
Create a table in your notebook similar to the one below to record
your date. Alternatively, there is a copy of this table on line that
you can print, cut out, and tape into your notebook.
2
Generate a set of five graphs, each showing BOTH the DNA and
BSA data generated from the Nucleic Acid program.
1 Unmixed DNA and unmixed BSA @ 100
2 DNA + BSA @ 100
-1
3 DNA + BSA @ 10
-2
4 DNA + BSA @ 10
-3
5 DNA + BSA @ 10
You can trace the graphs in order to superimpose them.
NanoDrop
Program
Sample
A260
DNA Control
BSA @ 10
0
BSA @ 10
-1
BSA @ 10
-2
BSA @ 10
-3
BSA @ 10
0
BSA @ 10
-1
BSA @ 10
-2
BSA @ 10
-3
DNA + BSA @
0
10
DNA + BSA @
-1
10
DNA + BSA @
-2
10
DNA + BSA @
-3
10
Nucleic Acid
Protein
Nucleic Acid
48
A280
Concentration
A260/A280

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