Mehmet Emre SUMER
Koc University-Molecular Biology and Genetics
17 DECEMBER 2010
Section 2- Group 5
MBGE 101 - Experiment 8:
Measuring DNA and Protein Concentration
Aim:
In this experiment, our aim is to estimate concentration and purity of given DNA and protein
samples. In order to determine concentrations, we use the UV absorbance of the samples at
wavelength of 260 nm and 280 nm.
Introduction:
There are many methods in molecular biology for determination DNA, RNA and protein
concentration of given samples. A common practice for obtaining qualitative and quantitative
information about those samples is to perform spectrophotometric analysis which means using
the UV absorbance at 260 nm and 280mnm. The UV method is very widely used even though it is
not highly accurate because it is a quick way, easy to apply, and little DNA is required.
Nucleic acids and proteins have different absorbance at wavelenght of 260 nm and 280 nm.
While proteins have little absorbance at 260 nm, both proteins and nucleic acids absorb UV light
at 280 nm. Therefore, if there is a sample which is mixture of protein an nucleic acid their
spectra overlap with each other, so it is not possible to determine their concentration. Indeed,
proteins have two absorbance peaks in the UV region. Because of the absorbtion by peptide
bonds there is one peak between 215-230 nm, and another peak at about 280 nm because of
aromatic amino acids (tyrosine, tryptophan, phenylalanine). Taking into account the nucleic
acids, there are actually two absorbance peaks depending on the subunit composition of DNA.
The purine subunits have an absorbance maximum slightly below 260 nm, and the pyrimidines
slightly above 260 nm. Therefore it is said that the absorbance peak of nucleic acids is at 260 nm.
Estimation of Concentration:
There are two UV methods for estimating concentration of pure samples. First method is
constructing a standard curve and the other is ‘short-cut’ method which is using the
absorptivity constants of proteins and nucleic acids. These absorptivity constants are calculated
by Beer-Lambert Law:
 A= e x l x c
A = absorbance
e = coefficient
l = pathlength
c = concentration
 c = A / (e x l)
Absorptivity constant of a protein varies depending on percentage of aromatic amino
acids it contains and absorptivity of DNA depending on its base composition and on whether it is
single-stranded or double-stranded. However, analysts commonly use ‘average’ absorptivity
constants . According to these average constants;



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 protein has an absorbance of 1 at 280 nm, then it contains
approximately 1 mg/mL of protein.
As a result;
 the concentration of a pure DNA
c = A260 x 50 x dilution ratio
 the concentraiton of a pure protein
c = A280 x 1 x dilution ratio
Estimation of Purity
To estimate the purity of a solution, the absorbance of the solution at two wavelengths,
260 nm and 280 nm, are measured and then the ratio of two absorbance calculated.



A260/A280 ratio of 2.0 is characteristic of pure RNA
A260/A280 ratio of 1.8 is characteristic of pure DNA
A260/A280 ratio of about 0.6 is characteristic of pure protein.
Materials:




UV spectrophotometer
Quartz cuvettes
DNA sample (1:40 diluted)
Bovine serum albumin (1:40 diluted)
Methods:

The absorbance of our samples 260 nm and 280 nm was meaured.

A fter that the A260/A280 ratio was calculated based on our measuruing.

Then, we determined whether the sample is DNA or protein based on the ratio.

We calculated the concentration of each sample according to their absorptivity
constants.
Results:
1
A260
2
3
4
5
6
7
8
0.123
0.156
0.278
0.352
1.110
0.091
0.280
0.207
0.103
0.160
0.321
0.225
0.735
0.049
0.180
0.157
1.194
0.975
0.866
1.564
1.51
1.857
1.56
protein
protein
protein
DNA
DNA
DNA
DNA
1.318
Protein
dominating
concentration 0.103
mg/ml
0.160
mg/ml
0.321
Mg/ml
0.018
Mg/ml
0.056
Mg/ml
0.005
Mg/ml
0.014
Mg/ml
0.010
Mg/ml
A280
A260/A280
Protein or
DNA
ε (for protein 280nm) 1.0 mg.cm/ml
For first sample
A= ε.l.c
A=0.103 l=1 cm ε=1.0 mg.cm/ml
A=1.0 l=1cm
c=A/ ε.l
ε(for DNA260 nm) 50μg.cm/Ml
A= ε.c.l
A=1.0 l=1cm
c=0.103/1cm.1.0mg.cm/ml
c=0.103 mg/ml
3.500
3.000
absorbation
2.500
2.000
quartz
1.500
plastic
1.000
0.500
0.000
-0.500
240
260
280
300
320
340
wavelength
Graph 1: absorbation difference for distilled water with quartz and plastic cuvettes
0.600
absorbation
0.500
0.400
0.300
DNA
protein
0.200
0.100
0.000
200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350
wavelength
Graph 2: DNA and protein absorbance in different wavelength
Discussion:
To understand which kind of cuvettes more appropriate, we use plastic and quartz cuvettes
with blank (distilled water) and we measured their absorbance. After measuring we obtained
absorbance for plastic cuvettes 3.000 and for quartz cuvettes 0,000. Based on these results we
can say quartz cuvettes are more appropriate to use. The reason that makes quartz cuvettes
more appropriate is ability of transparence throughout the UV completely.
When we try to decide which one is DNA or protein, we looked their absorbance ratio. Because
we know that when the absorbance ratio is close to 0.6, it means that the sample is protein. On
the other hand, when the absorbance ratio is close to 1.8, it means that the sample is DNA.
Questions:
1.Examine your four absorbance spectra. Do the spectra overlap? If so, at what
wavelength(s)? Compare the spectrum of the mixture of nucleic acids and proteins
to the pure samples. What features of the spectra vary? Where do they overlap?
Yes, the spectra do overlap in 268 wavelength. We find the overlap for mixture by
summing up absorbance of DNA and protein. Higher value of this summing shows peak
point and if there is a peak point , spectra do overlap .
2. Fill in this table.
UV SPECTROPHOTOMETRY OF DNA AND PROTEINS
DNA
A260
: 0,358
A280
: 0,21
A260/A280
: 1,705
Purity?
: impure
Concentration : 0.0105
PROTEIN
A260
: 0.231
A280
: 0,318
A260/A280
: 0,736
Purity?
: impure
Concentration :0,318
MIXTURE
A260
:0.207
A280
:0,157
A260/A280
:1.318
Purity?
:DNA dominating
Concentration :0,010
3. These UV assays are very commonly used by molecular biologists. Why? What
information is obtained from these simple assays?
This method generally is used by molecular biologist because molecular biologist work
with small molecules such as DNA, RNA, nucleic acids and this method is very good to
ubderstand concentration of these small molecules.
4. What are the assumptions of these UV methods?
UV methods is very good method to understand absorbance and concentrations of small
molecules as standard absorbation method . Furthermore ,Different small molecules
weight products and their absorbance can be calculated by this method. Another
important asumption of these methods is wavelengths. this assumption is also very
importan because with this assumption, purity and concentration of DNA, RNA, and
protein are calculated .
5. 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.
The UV methods is widely used because it is a quick and easy way to estimate purity and
concentration of DNA and protein. In addition, it requires little amounts of samples and
lower cost than the other methods, however it is not highly accurate. For example, at
280 nm both proteins and nucleic acids can absorb light. Therefore, if we have a sample
which is mixture of nucleic acid and protein it is hard to determine purity and
concentration of the sample. At 260 nm, there is another problem. While we are
performing the UV method with a DNA sample, we ignore the absorption difference
between purine and pyrimidine subunits.There are some assumptions used not to
handle with these problems. The average absorptivity constant is one of these
assumptions. The operations done by using these constants do not give the actual
results. Taking into account the purity, the UV method do not actually seperate DNA and
RNA form each other.On the other hand, to form a general idea about given samples the
UV method is the simplest and the quickest way.
REFERENCES:
-
Glasel, J.A. (1995) Validity of Nucleic Acid Purities Monitored by A260/A280
Absorbance Ratios, Biotechniques 18:62-63.
http://www.biotek.com/resources/articles/nucleic-acid-purity-a260a280.html