find the concentration of the sample. Concentration and quality of
a sample of DNA are measured with a UV spectrophotometer. A
standard graph can be drawn using various concentrations of DNA
and OD (optical density) values.
4.2.1 Materials Required
l
DNA sample.
l
Tris–EDTA (TE) buffer.
l
UV spectrophotometer.
4.2.2 Procedure
1. Add 10 μl DNA sample in TE buffer.
2. Dilute the above DNA sample by the factor of 100, i.e., by
taking 10 μl of the sample in 990 μl of TE buffer.
3. Measure optical density value at A260 and A280 and calculate
the amount of DNA.
4. Use the following formula to determine the concentration of
DNA:
Total DNA (μg) ¼ (A260) (50 μg/ml/A260) (100)
(0.1 ml),
where 100 is the dilution factor.
0.1 ml is the total volume of the DNA.
4.3 DNA Quality
Measurement
DNA quality identification is based on the fact that OD at 260 nm
is twice than at 280 nm if the solution comprises pure DNA. If
there is a impurity, there is some additional OD, which drops the
OD ratio between 260 and 280 nm. Clean DNA has an OD260/
OD280 between 1.8 and 2.0.
4.4 Agarose Gel
Electrophoresis
Agarose gel electrophoresis is used to examine and quantify nucleic
acid. Agarose is a linear polymer made up of repeating units of
1,3-linked ß-D-galactopyranose and 1,4-linked 3,6-anhydro-α-Lgalactopyranose [P-D-gal (1-4)-3,6-anhydro-α-L-Gal (1-3)-]n.
Agarose has an approximate MW of 12,000 and comprises about
35–40 agarobiose units. Agarose in solution exist as left-handed
double helices. About 7–11 such helices form bundles which
lengthen as long rods and appear to intertwine with one another,
further firming up the frame work of the gel. The cross links are
apprehended together by hydrogen and hydrophobic bonds. By
shifting the gel concentration, the pre-size can be changed. Higher
the concentration of agarose, smaller the pre-size and vice versa.
Due to large pore size even at low concentration, agarose gels are
extensively used for the separation of DNA and RNA.
4.4.1 Materials Required
l
Preparation of reagent.
Molecular Characterization
135
a sample of DNA are measured with a UV spectrophotometer. A
standard graph can be drawn using various concentrations of DNA
and OD (optical density) values.
4.2.1 Materials Required
l
DNA sample.
l
Tris–EDTA (TE) buffer.
l
UV spectrophotometer.
4.2.2 Procedure
1. Add 10 μl DNA sample in TE buffer.
2. Dilute the above DNA sample by the factor of 100, i.e., by
taking 10 μl of the sample in 990 μl of TE buffer.
3. Measure optical density value at A260 and A280 and calculate
the amount of DNA.
4. Use the following formula to determine the concentration of
DNA:
Total DNA (μg) ¼ (A260) (50 μg/ml/A260) (100)
(0.1 ml),
where 100 is the dilution factor.
0.1 ml is the total volume of the DNA.
4.3 DNA Quality
Measurement
DNA quality identification is based on the fact that OD at 260 nm
is twice than at 280 nm if the solution comprises pure DNA. If
there is a impurity, there is some additional OD, which drops the
OD ratio between 260 and 280 nm. Clean DNA has an OD260/
OD280 between 1.8 and 2.0.
4.4 Agarose Gel
Electrophoresis
Agarose gel electrophoresis is used to examine and quantify nucleic
acid. Agarose is a linear polymer made up of repeating units of
1,3-linked ß-D-galactopyranose and 1,4-linked 3,6-anhydro-α-Lgalactopyranose [P-D-gal (1-4)-3,6-anhydro-α-L-Gal (1-3)-]n.
Agarose has an approximate MW of 12,000 and comprises about
35–40 agarobiose units. Agarose in solution exist as left-handed
double helices. About 7–11 such helices form bundles which
lengthen as long rods and appear to intertwine with one another,
further firming up the frame work of the gel. The cross links are
apprehended together by hydrogen and hydrophobic bonds. By
shifting the gel concentration, the pre-size can be changed. Higher
the concentration of agarose, smaller the pre-size and vice versa.
Due to large pore size even at low concentration, agarose gels are
extensively used for the separation of DNA and RNA.
4.4.1 Materials Required
l
Preparation of reagent.
Molecular Characterization
135