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Sunday, December 13, 2009

HOW TO ESTIMATE CONTENT OF DNA IN A GIVEN SAMPLE

How to determine the DNA content of given sample by diphenylamine reaction.

When DNA is treated with diphenylamine acidic conditions a blue compound is formed with sharp absorption maxima at 595 nm. This reaction is given by 2’- deoxy pentose in general and is not specific for DNA. In acid soln the straight chain form of deoxy ribose is converted to highly reactive &-hydroxyl laevulin aldehyde which reacts with biphenyl amine to give blue columned complex. In DNA only the deoxyribose of Purina nucleotide reacts so that the value obtained represents half of the total deoxyribose present.
Requirement: - 1) std. DNA soln (1 mg/ml)
2) Diphenylamine reagent

Procedure: - To 1 ml of sample and std. add 2 ml water add 5 ml of diphenylamine reagent. Heat the soln on a water bath for 10 mins. and cool measure absorbance at 595 nm against water blank.


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