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spectrophotomet
ry and
electrophoresis
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Q)Numerate the methods that are used to
?guantifyinq of DNA
Spectrophotometry:
Q)What is Spectrophotometric Instruments?
A spectrophotometer is used to measure the light
transmitted by a solution to determine the concentration
of the light-absorbing substance in the solution.
A = cl
Where A=absorbance, =extinction coefficient,
c=concentration and l=path length.
The BeerLambert law draws a direct correlation between
absorbance and concentration.
Q)What are the principle of DNA quantification
using NanoDrop?
While nucleic acids absorb at many wavelengths, they
have a peak absorbance of UV light at260nm. Thus, the
amount of light absorbed in this region can be used to
determine the concentration of RNA or DNA in solution by
applying the BeerLambert law. For DNA, absorbance at
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A260 (also called optical density, OD) is converted into
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spectrometer utilizing a linear CCD array is used to analyze
the light after passing through the sample. The instrument
is controlled by PC based software, and the data is logged
in an archive file on the PC.
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Q) Why does DNA absorb light in the UV light
region at260nm?
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? Q)What is TRIzol
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c. To degrade mitochondria
d. To limit the amount of magnesium salts in the lysate
e. To destroy the three dimensional structure of proteins
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bromide-stained DNA after agarose minigel
electrophoresis
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nm) and purified proteins(280 nm). This would include
dsDNA, ssDNA, RNA and purified proteins.
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Agarose gel electrophoresis method
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Schematic illustration of a typical horizontal gel
electrophoresis setup for the separation of nucleic
acids.
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2. Voltage:The higher the voltage, the faster the DNA
moves. But voltage is limited by the fact that it heats and
ultimately causes the gel to melt. High voltages also
decrease the resolution (above about 5 to 8 V/cm)
3. Agarose
4. Buffer
5. Visualization
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The disadvantage of higher concentrations is the long run
times (sometimes days).
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C. SB: Sodium borate.
TAE the best one it has the lowest buffering capacity but
provides the best resolution for larger DNA. This means a
lower voltage and more time, but a better product.
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Silver staining (SS) :Silver staining is a highly sensitive
method for the visualization of nucleic acid and protein
bands after electrophoretic separation on polyacrylamide
gels. Nucleic acids and proteins bind silver ions, which can
be reduced to insoluble silver metal granules. Sufficient
deposition is visible as a dark brown band on the gel.
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(Chevallet et al., 2006).
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