BACKGROUND
The basic objective of this experiment is to determine the concentration of DNA by utilizing diphenylamine method.
Principle:
The principle behind this experiment is the reaction between diphenylamine and deoxyribose sugar that produces blue colour complex. The DNA sample when boiled under acidic conditions, it results in depurintion of DNA which is followed by dehydration of deoxyribose sugar into highly reactive w-hydroxylevulinylaldehyde.
This in turn under acidic conditions reacts with diphenylamine to make a blue coloured complex that absorbs at 595 nm.The sugar that is linked to purine residues takes part in the reaction therefore the readout is only from 50% of the total nucleotides. This holds true for both known standard and the given unknown sample therefore the concentration of the unknown sample is directly calculated from the standard graph
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REQUIREMENTS
Reagents:
Diphenylamine reagent- In a 250 m coloured glass bottle take 1g diphenylamine. To it add 100 ml glacial acetic acid and mix it well so that it dissolves completely. Then add 2.5 ml of concentrated sulphuric acid. The reagent should be stored in dark at a temperature 2-8oC.
Calf thymus DNA- Make 100 microgram of calf thymus DNA solution in distilled water.
Glacial acetic acid
Concentrated sulphuric acid
Others:
A UV/Visible spectrophotometer
Vortex mixer
Weighing balance
Water bath
Pipettes
Pipette tips
A 5 ml glass pipette
Pipette aid
A 100 ml measuring cylinder
A 250 ml amber coloured glass bottle
Test tubes
Caps for glass tubes
Distilled water
Quartz or glass cuvettes
PROCEDURE
The assay is done with range of dilutions (1, 1:10, 1:100 and 1:1000). Make 1 ml of each of these dilutions. Bring 15 test tubes and label them 1-15.1 Then with help of pipette take out 100 µl, 200 µl, 300 µl…1000 µl calf thymus DNA standard in the tubes that is labelled 1-10. In the tube 11 put distilled water that acts as blank. In each tube now add distilled water to make the total volume 1 ml.
Now in the tube label 12-15 put 1 ml of unknown DNA solutions. Now add 3 ml of DPA reagent in every tubes and mix it well by vortexing.2 Now cover the tube with caps and keep them in boiling water for about 10 minutes. After 10 minutes bring out all the tubes from the water bath and allow them to come down to room temperature. Measure the absorbance at 1-10 and 12-15 at 595 nm in the glass cuvette against the blank.
Now calculate the concentration of DNA.3 Plot a graph with absorbance values of tubes 1-10 against the standard DNA amount that is added in the tube. Now fit the data points by using linear regression. Estimate the slope (difference in absorbance/difference in DNA amount).4
CONCLUSION
This test is very specific to determine the concentration of DNA and is widely used.
REFERENCES
- Bartley W. Efficiency of oxidative phosphorylation during the oxidation of pyruvate. Biochem J. 1953 Jul; 54(4):677–682.
- Berenblum I, Chain E. An improved method for the colorimetric determination of phosphate. Biochem J. 1938 Feb; 32(2):295–298.
- Burton K. The relation between the synthesis of deoxyribonucleic acid and the synthesis of protein in the multiplication of bacteriophage T2. Biochem J. 1955 Nov;61(3)
- Ceriotti G. A microchemical determination of deoxyribonucleic acid. J Biol Chem. 1952 Sep; 198(1):297–303.
Also read:
- Isolation, screening and purification of an unknown organism from soil
- Cell Immobilization Techniques
- Fermentation process of wine production
- Fermentation process of alcohol production
- Determination of saponification value of the given oil/fat
- Determination of saponification value of fat (coconut oil)
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