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Dna Estimation by Dpa Method

This document describes a method to estimate the concentration of DNA using the diphenylamine reaction. DNA is treated with acid to produce an aldehyde, which reacts with diphenylamine to form a blue complex measurable at 595 nm. Standard DNA solutions are prepared and treated with diphenylamine reagent before reading absorbance. A standard curve of absorbance versus concentration is made to determine the unknown DNA concentration from its absorbance.

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Praveen Roylawar
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2K views1 page

Dna Estimation by Dpa Method

This document describes a method to estimate the concentration of DNA using the diphenylamine reaction. DNA is treated with acid to produce an aldehyde, which reacts with diphenylamine to form a blue complex measurable at 595 nm. Standard DNA solutions are prepared and treated with diphenylamine reagent before reading absorbance. A standard curve of absorbance versus concentration is made to determine the unknown DNA concentration from its absorbance.

Uploaded by

Praveen Roylawar
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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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Estimation of DNA by Diphenylamine method

Dr. Mahesha H B, Yuvaraja’s College, Mysore.


Aim: To estimate the concentration of DNA by diphenylamine reaction.
Principle: This is a general reaction given by deoxypentoses. The 2-deoxyribose of DNA, in the presence of
acid, is converted to ω-hydroxilevulinic aldehyde, which reacts with diphenylamine to form a blue coloured
complex, which can be read at 595 nm.
Requirements:
1. Standard DNA solution- Dissolve calf thymus DNA (200µg/ml) in 1N perchloric acid/buffered saline.
2. Diphenylamine solution- Dissolve 1g of diphenylamine in 100 ml of glacial acetic acid and 2.5 ml of
concentrated H2SO4. This solution must be prepared fresh
3. Buffered Saline- 0.5 mol/litre NaCl; 0.015 mol/litre sodium citrate, pH 7.
Procedure:
1. Pipette out 0.0, 0.2, 0.4, 0.6, 0.8 and 1 ml of working standard in to the series of labeled test tubes.
2. Pipette out 1 ml of the given sample in another test tube.
3. Make up the volume to 1 ml in all the test tubes. A tube with 1 ml of distilled water serves as the blank.
4. Now add 2 ml of DPA reagent to all the test tubes including the test tubes labeled 'blank' and 'unknown'.
5. Mix the contents of the tubes by vortexing / shaking the tubes and incubate on a boiling water bath for 10
min.
6. Then cool the contents and record the absorbance at 595 nm against blank.
7. Then plot the standard curve by taking concentration of DNA along X-axis and absorbance at 595 nm
along Y-axis.
8. Then from this standard curve calculate the concentration of DNA in the given sample.
Result: The given unknown sample contains ----µg DNA/ml.
Observations and Calculations
Volume of Volume Concent Volume
standard of ration of of DPA Incuba
(200 distilled DNA reagent te A595
µg/ml) water (µg) (ml) in
DNA (ml) (ml)
boiling
water
0.0 1.0 00 2 0.00
bath
0.2 0.8 40 2 for
0.4 0.6 80 2 10
Min
0.6 0.4 120 2
&
0.8 0.2 160 2 Cool
1.0 0.0 200 2
1.0 0.0 To be 2
Unknown Estimate
d
----------------

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