Estimation of DNA
Aim
To estimate the amount of DNA in the given sample
Principle
Colorimetric determination of DNA concentration is done by diphenylamine reaction. This procedure involves chemical hydrolysis of DNA during which the glycosidic bond linking the base to the sugar is hydrolyzed with subsequent hydrolysis of the phosphodiester bond linking that nucleotide to the next in a polynucleotide chain. The reaction requires a deoxyribose sugar and therefore is specific for DNA. Purine nucleotides are more labile to these hydrolysis conditions than pyrimidine nucleotides, but eventually complete hydrolysis can occur. Under these conditions, the 2-deoxyribose is converted to hydroxylevulinyl aldehyde, which reacts with the compound, diphenylamine, to produce a blue-colored compound. The amount the blue-colored compound formed is determined by measuring the intensity of absorbance of the solution at 595 nm. This is related to the DNA concentration by constructing a calibration curve, which gives the exact relationship between A595 and DNA concentration.
Materials required
1. Diphenyl amine reagent – 1 gm of diphenyl amine in 100 ml glacial acetic acid and 2.5 ml of concentrated sulfuric acid.
2. Standard DNA solution – 250 µg DNA/ ml of distilled water
Procedure
1. 0.2, 0.4, 0.6, 0.8 and 1 ml of Standard DNA solution was pipetted out into a series of test tubes.
2. 1 ml of the unknown or sample DNA solution was pipetted out into another test tube.
3. The volume was made up to 1 ml in all test tubes using distilled water. A tube with 1 ml distilled water served as the blank.
4. 2 ml of diphenyl amine reagent was added.
5. The mixture was heated in a boiling water bath for 10 minutes and cooled at room temperature.
6. The deep blue colour developed was measured at 595 nm against blank.
7. A standard graph was plotted and amount of DNA in the given sample was calculated.
Observation
Sl No.
|
Volume of DNA solution (ml)
|
Volume of distilled water (ml)
|
Volume of diphenyl amine reagent(ml)
|
Concentration of DNA (µg)
|
Optical density at 595 nm
|
1
|
0.0
|
1.0
|
2.0
|
0
|
……..
|
2
|
0.2
|
0.8
|
2.0
|
50
|
……
|
3
|
0.4
|
0.6
|
2.0
|
100
|
…….
|
4
|
0.6
|
0.4
|
2.0
|
150
|
…….
|
5
|
0.8
|
0.2
|
2.0
|
200
|
……
|
6
|
1.0
|
0.0
|
2.0
|
250
|
…….
|
7
|
1.0
|
0.0
|
2.0
|
……
|
…...
|
Calculation
From graph, concentration of the given sample is
By calculation
OD of the test = Concentration of test
OD of standard Concentration of standard
concentration of the given sample is
Result
The amount of DNA in the given sample
From graph =
From calculation =
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