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DNA quality

spectrophotomet
ry and
electrophoresis

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Q)Numerate the methods that are used to
?guantifyinq of DNA

The most commonly used methodologies for quantifying


:the amount of nucleic acid in a preparation are
electrophoretic run along with standard DNA (A)
spectrophotometric estimation (DNA quantification (B)
using NanoDrop)
flourometric determination (C)

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.

Q)The BeerLambert Lawwhat does it mean?

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

DNA concentration by following method:


A260/OD of 1.0 = a concentration of 50 g/ml

of double-stranded DNA (dsDNA)


DNA concentration (g/ml): A260 X 50
:DNA yield
DNA conc. X Total volume of DNA
solution
DNA purity: A260/A280 ratio: 1.7 1.9
Describe NanoDrop Instrument?
Instrument Description NanoDrop is Spectrophotometer
measures 1 ul samples with high accuracy and
reproducibility. The full spectrum (220nm-750nm)
spectrophotometer utilizes a patented sample retention
technology that employs surface tension alone to hold the
sample in place. This eliminates the need for cumbersome
cuvettes and other sample containment devices and allows
for clean up in seconds. In addition, the NanoDrop
Spectrophotometer has the capability to measure highly
concentrated samples without dilution (50X higher
concentration than the samples measured by a standard
cuvette spectrophotometer).

Q)How can NanoDrop Operate?


A 1 ul sample is pipetted onto the end of a fiber optic cable
(the receiving fiber). A second fiber optic cable (the source
fiber) is then brought into contact with the liquid sample
causing the liquid to bridge the gap between the fiber optic
ends. The gap is controlled to both 1mm and 0.2 mm paths.
A pulsed xenon flash lamp provides the light source and a

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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?

presence of double bond system in the purine and


pyrimidine bases makes DNA absorb light in the UV light
region at260nm. Nucleic acids absorb UV light at 260 nm
due to the aromatic base moieties within their
structure. Purines (thymine, cytosine and uracil) and
pyrimidines (adenine and guanine) both have peak
absorbances at 260 nm, thus making it the standard for
quantitating nucleic acid samples.

Q) What does Absorbance at 280 nm mean?


The 280 nm absorbance is measured because this is
typically where proteins and phenolic compounds have a
strong absorbance. Aromatic amino acid side chains
(tryptophan, phenylalanine, tyrosine and histidine) within
proteins are responsible for this absorbance. Similarly, the
aromaticity of phenol groups of organic compounds
.absorbs strongly near280 nm

Q)What does Absorbance at 230 nm mean?


Many organic compounds have strong absorbances at
around 225 nm. In addition to phenol, TRIzol, and
chaotropic salts, the peptide bonds in proteins absorb light
between 200 and 230 nm.

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? Q)What is TRIzol

rand name of a chemical solution used in RNA/DNA/protein


extraction, by the reference paper from TRIzol. The
correct name of the method is guanidinium thiocyanate-
phenol-chloroform extraction. The use of TRIzol can result
in DNA and RNA yields comparable to other extraction
methods. TRIzol is light sensitive and is often stored in a
dark-colored, glass container covered in foil. It must be
.kept below room temperature

Q)What does A chaotropic agent mean and why it is


?used in DNA isolation

A chaotropic agent is a molecule in water solution that can


disrupt the hydrogen bonding network between water
molecules. This has an effect in the stability of the native
state of other molecules in the solution, mainly
macromolecules (proteins, nucleic acids) by weakening
the hydrophobic effect. For example, a chaotropic agent
reduces the amount of order in the structure of a protein
formed by water molecules, both in the bulk and the
hydration shells around hydrophobic amino acids, and
may cause its denaturation.
A chaotropic agent is often used in DNA isolation. Its
purpose is
a. To degrade membrane lipids
b. To destroy the endoplasmic reticulum

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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

Q)What does A260/280 ratio mean?

The A260/280 ratio is generally used to determine protein


contamination of a nucleic acid sample. The aromatic
proteins have a strong UV absorbance at 280 nm. For pure
RNA and DNA, A260/280 ratios should be somewhere
around 2.1 and 1.8, respectively. A lower ratio indicates
the sample is protein contaminated. The presence of
protein contamination may have an effect on downstream
applications that use the nucleic acid samples.

Q)What does A260/230 ratio mean?

The A260/230 ratio indicates the presence of organic


contaminants, such as (but not limited to): phenol, TRIzol,
chaotropic salts and other aromatic compounds. Samples
with 260/230 ratios below 1.8 are considered to have a
significant amount of these contaminants that will
interfere with downstream applications. This is especially
true for reverse transcription. In a pure sample, the
A260/230 should be close to 2.0
Q)What are the disadvantages of this method?
1. does not work with heavily contaminated DNA
2. cannot be used if only small amounts of DNA are
available; better suited for small amounts and more
reliable in general is the quantification of ethidium

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bromide-stained DNA after agarose minigel
electrophoresis

Q) What are the sample size requirements for the


NanoDrop?
A: Although 1 ul volumes are usually sufficient for most
sample measurements, increasing the sample size to 2 ul
will ensure proper column formation for samples with
reduced surface tension properties.

Q: Will the sample size affect the concentration


results?
A: No. All calculations are volume independent. Sample
concentrations for all applications are calculated using the
Beer-Lambert equation, which relates concentration to
absorbance using analyte and wavelength specific
extinction coefficients or conversion factors.

Q: What pathlengths are used to make


measurements and is the user required to make
any calculations relevant to the pathlength?
A: The NanoDrop uses a 0.5 mm pathlength and all
reported concentration results have taken into account
the pathlength. The absorbance reported for all
measurements is normalized to a 10 mm pathlength.

Q: What types of samples can be measured with the


NanoDrop?
A: The NanoDrop is designed to measure the absorbance
and calculate the concentration of nucleic acids (260

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nm) and purified proteins(280 nm). This would include
dsDNA, ssDNA, RNA and purified proteins.

Q: Do nucleic acids require purification prior to


measurement?
A: Yes. Absorbance measurements are not specific for a
particular sample type. Any analyte that absorbs at 260
nm (DNA, RNA or free nucleotides) will contribute to the
total absorbance of the sample.

Q)What is Agarose gel electrophoresis (AGE) based


on?

Agarose gel electrophoresis is a quick and easy molecular


technique used to analyze and separate nucleic acids
based on their size (i.e. how many base pairs a molecule is
composed of). Electrophoresis takes advantage of the fact
that DNAs phosphate backbone is negatively charged.
Thus when DNA is placed in an electric field, it will migrate
toward the positive electrode. The differential ability of
DNA to move through a gel based on its size doesnt really
depend on the electric field or the charged properties of
DNA, but more importantly on the composition of the gel.

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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.

Q)What is Agarose gel?


Agarose is a natural linear polymer extracted from
seaweed that forms a gel matrix by hydrogen-bonding
when heated in a buffer and allowed to cool. Agarose gel
electrophoresis can be used for the separation of DNA
fragments ranging from 50 base pair to several
megabases (millions of bases) using specialized
apparatus.

What are the Factors that affecting DNA Migration?

1. DNA or RNA Molecular Weight: The length of the


DNA molecule is the most important factor, smaller
molecules travel farther.

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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

What the concentration of Agarose gel is


recommended?
The concentration of agarose is referred to as a
percentage of agarose to
volume of buffer (w/v), and agarose gels are normally in
the range of 0.2% to 3% .
What does the lower the concentration of agarose
gel mean?
The lower the concentration of agarose, the faster the
DNA fragments migrate. In general, if the aim is to
separate large DNA fragments because they result in
greater separation between bands that are close in size., a
low concentration of agarose should be used.

What does the higher the concentration of agarose


gel mean?
If the aim is to separate small DNA fragments, a high
concentration of agarose is recommended. Increasing the
agarose concentration of a gel reduces the migration
speed and enables separation of smaller DNA molecules.

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The disadvantage of higher concentrations is the long run
times (sometimes days).

Most agarose gels:


1. 1% gels are common for many applications.
2. 0.7%: good separation or resolution of large 510kb
DNA fragments
3: 2% good resolution for small 0.21kb fragments.
4: Up to 3% can be used for separating very tiny
fragments but a vertical
polyacrylamide gel is more appropriate in this case.

What are the properties of Electrophoretic buffer


systems?
Effective separation of nucleic acids by agarose or
polyacrylamide gel electrophoresis depends upon the
effective maintenance of pH within the matrix. Therefore,
buffers are an integral part of any electrophoresis
technique. Moreover, the electrophoretic mobility of DNA
is affected by the composition and ionic strength (salt
content) of the electrophoresis buffer Without salt,
electrical conductance is minimal and DNA barely moves.
In a buffer of high ionic strength, electrical conductance is
very efficient and a significant amount of heat is
generated.

What are the most common buffers for agarose gel


and which are the best one?
A. TAE: tris acetate EDTA
B. TBE: Tris/Borate/EDTA

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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.

What are the Advantages of agarose gel


electrophoresis?
1. Nontoxic gel medium
2. Gels are quick and easy to cast
3. Good for separating large DNA molecules
4. Can recover samples by melting the gel,
5. digesting with enzyme agarose or treatingwith
chaotropic salts

What are the disadvantages of agarose gel


electrophoresis?
1. High cost of agarose
2. Fuzzy bands
3. Poor separation of low molecular weight samples.

How we can visualizing the DNA bands in agarose


gel electrophoresis?
After the electrophoresis has been completed there are
different methods that may be used to make the
separated DNA species in the gel visible to the human
eye. Ethidium bromide staining (EBS) the
localization of DNA within the agarose gel can be
determined directly by staining with low concentrations
of intercalating fluorescent ethidium bromide dye under
ultraviolet light.

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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.

development prior to excessive background formation and


to remove excess silver ion

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(Chevallet et al., 2006).

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