Professional Documents
Culture Documents
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
Hana Hakami
2011
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
Hana Hakami
2011