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Estimation of DNA by DPA

This document describes a method for estimating DNA concentration using a diphenylamine reaction. DNA is reacted with diphenylamine and acid to produce a blue compound with maximum absorption at 595nm. Standard DNA solutions are used to generate a calibration curve relating absorbance to DNA concentration. Unknown DNA sample absorbances are then used to determine concentration from the standard curve. The procedure involves preparing a diphenylamine reagent, setting up standard and sample test tubes, incubating the reactions, measuring absorbances, and calculating concentrations from the standard curve.

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148 views2 pages

Estimation of DNA by DPA

This document describes a method for estimating DNA concentration using a diphenylamine reaction. DNA is reacted with diphenylamine and acid to produce a blue compound with maximum absorption at 595nm. Standard DNA solutions are used to generate a calibration curve relating absorbance to DNA concentration. Unknown DNA sample absorbances are then used to determine concentration from the standard curve. The procedure involves preparing a diphenylamine reagent, setting up standard and sample test tubes, incubating the reactions, measuring absorbances, and calculating concentrations from the standard curve.

Uploaded by

itschandansarma
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© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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BT 510 Analytical Biotechnology Lab

ESTIMATION OF DNA BY DIPHENYLAMINE REACTION

Principle:
The deoxyribose in DNA in the presence of acid forms β-hydroxylevulinaldehyde which
reacts with diphenylamine to give a blue colour with a sharp absorption maximum at
595nm. In DNA, only the deoxyribose of the purine nucleotides react, so that the value
obtained represents half of the total deoxyribose present.

METHODOLOGY

a) Materials required:

1. Equipments:
• Spectrophotometer
• Water bath

2. Chemicals/reagents:
• Standard DNA solution (0.25mg/ml)
• Diphenylamine reagent
• DNA sample in saline citrate buffer
• Saline citrate buffer (0.15M NaCl, 0.015M sodium citrate, pH 7.0)
• Glacial acetic Acid
• Concentrated H2SO4
• Ethanal

3. Glasswares and others:


• Test tubes
• Pipettes
• Graduated cylinder

b) Procedure:

Preparation of reagent: Dissolve 1.5g diphenylamine in 100ml of glacial acetic acid.


Add 1.5ml of conc H2SO4. Store the solution in a dark glass bottle. On the day of use,
prepare a fresh solution of ethanal (1ml) in dH2O (50ml). Add 0.5ml of this solution to
each 100ml of the diphenylamine solution.
Caution: Wear eye protection and use a fume cupboard when preparing this reagent.
Diphenylamine is harmful if ingested or inhaled and may irritate skin or eyes if it
comes into contact with them.
c) Assay:

1. Prepare a series of dilutions of standard DNA (0.25mg/ml) in saline citrate buffer


to give a concentration of 50-500µg/ml.
2. Prepare all the samples in triplicate.
3. To 2ml of each dilution of blank, standard and unknown add 4ml of
diphenylamine reagent and mix. Tube1 is used as blank and tubes 2 through 7 are
used for construction of a standard calibration curve for DNA. Tubes 8-11 are for
unknown samples. (Table1)
4. Incubate all the tubes in boiling water for 10 min.
5. Cool the tubes and read the absorbance at 595nm against the blank.
6. Construct a standard curve of absorbance A595 vs. quantity of DNA and then
calculate the concentration of unknown DNA dissolved in the saline citrate
solution.
Table1:
Sl DNA DH2O Reagent A595
(µl) (ml)
No (µl) (µg)
1. - - 2000 4
2. 200 50 1800 4
3. 400 100 1600 4
4. 800 200 1200 4
5. 1200 300 800 4
6. 1600 400 400 4
7. 2000 500 - 4
8. Unknown (A) - - 4
9. Unknown (B) - - 4
10. Unknown (C) - - 4
11. Unknown (D) - - 4

Calculation: Determine the amount of DNA in the unknown sample by plotting a standard
curve of A595 on Y-axis and µg of DNA on X-axis.
References:
1. Plummer, D.T. (1977) An Introduction to Practical Biochemistry. Tata McGraw Hill,
Bombay.
2. J. Jayaram. (1981) Laboratory Manual in Biochemistry. New Age International Ltd.
New Delhi.
3. Burton, K. (1956) A study of the conditions and mechanism of the diphenylamine
reaction for the colorimetric estimation of deoxyribonucleic acid. Biochemical Journal.
62: 314-323.

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