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Hana Hakami

2011

DNA Quantification
Introduction
When working with DNA, it is important to know its
concentration. The easiest means of determining DNA
concentration is through spectrophotometric analysis. The
concentration of DNA will be expressed in units of mass
per volume; for example: ng/ml or g/ml.
First: Detremination of DNA Concentration
There are more types of spectrophotometer and
cuvette, but we will use GeneQuamt spectrophotometer
with quartz cuvette, and NanoDrop Spectrophotometer.
Principle
Nucleic acids, like many other substances, have the
property of absorbing light at a specific wavelength. Since
nitrogenous bases absorb UV light, the more concentrated
the DNA solution, the more UV light it will absorb.
DNA and RNA absorb light maximally at a wavelength
of 260 nm.
Because of this property, they can be
quantified spectrophotometrically with UV light source.
The concentration of pure double-stranded DNA with
an A260 of 1.0 is 50 g/ml. In order to there is a linear
relationship between absorbance and DNA concentration,
we can use Thus, one can use the following formula to
determine the DNA concentration of a solution:
Unknown g/ml = 50 g/ml x Measured A260 x dilution
factor

The purity of a nucleic acid solution can be


determined by calculating the A260/A280 ratio.
The
nucleic acid absorbs maximally at 260 nm and protein (a
principle contaminant) absorbs maximally at 280 nm.
Pure DNA has an A260/A280 ratio of 1.8.

Hana Hakami

2011

Pure RNA has an A260/A280 ratio of 2.0.

Cuvettes may be made of plastic, glass, crystal or


quartz. The choice of cuvette depends on the wavelength
at which the absorbance is to be measured. Glass or
plastic cuvettes can be used at wavelengths greater than
340 nm whereas quartz cuvettes are used in the
ultraviolet range because plastic and glass absorb UV
light.
1.
Determination
of
DNA
GeneQuamt spectrophotometer

concentration

by

Materials
Spectrophotometer - Quartz Cuvette - Disttelted Water TE Buffer - DNA Templates - Microfuge tubes - Micro
pipettes - Tips - Gloves Biohazard Bags & Container
Protocol
1. Prepare your blank in microfuge tube as following:
98 l d.d.H2O + 2 l DNA Hydration Solution (TE Buffer).
2. Prepare your DNA samples in microfuge tubes as
following:
98 l d.d.H2O + 2 l DNA Template.
3. Mix them well.
4. Turn on the Spectrophotometer (chose 50 as dilution
factor-why?).
5. Transfer the blank to Cuvette and read blank.
6. Now, after blank wash the cuvette by d.d.H2O
7. Transfer the first sample to cuvette and read sample.
8. Record the reading of DNA concentration and the
ratio of A260/A280.
9. Wash the cuvette with d.d.H2O .
10.
Repeat steps number 7, 8 and 9 for every
sample.
2.
Determination
of
DNA
NanoDrop Spectrophotometer
Materials

concentration

by

Hana Hakami

2011

NanoDrop Spectrophotometer - d.d.H2O - TE Buffer - DNA


Templates Kimwipes Tissue - Micro pipettes - Gloves
Biohazard Bags & Container
Protocol
1. Turn on the NanoDrop Spectrophotometer and its
software.
2. Open NanoDrop program and press on nuclic acids
button.
3. Clean the surface of NanoDrop spectrophotometer by
use d.d.H2O
4. Add your blank as following:
1 l DNA Hydration Solution (TE Buffer).
5. Read the blank by press on Blank button.
6. Clean the surface of NanoDrop spectrophotometer by
use d.d.H2O
7. Add the first DNA sample as following:
1 l DNA Template.
8. Read the concentration of DNA by press on Measure
button.
9. Clean the surface of NanoDrop spectrophotometer by
use d.d.H2O
10.
Repeat steps number 7, 8 and 9 for every
sample.
11.
Take the readings of DNA concentrations.

Second: Preparation of Working DNA


When working with DNA in any biotechnology as PCR,
you must to use same concentration for all DNA samples.
The DNA on this concentration is called Working DNA.
Choice of working DNA concentration depends on many
factors like, the concentrations of DNA stock, type of
biotechnique and experience of searcher.
We will choose 50 g/ml or 25 g/ml as working DNA
concentration by use the following formula:
C1 x V 1 = C 2 x V 2

Hana Hakami

2011