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  Section: Plant Lab Protocols
 
 
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Methodology for Nucleic Acids

 
     
 
Estimation of DNA

Estimation of DNA is possible by a number of methods based on the physical or chemical property of the nucleic acid. A convenient and easy colorimetric method is available on the basis of quantitative reaction of deoxysugar with diphenylamine reagent.
 
 



Principle

Under extreme acid conditions, DNA is initially depurinated quantitatively followed by the dehydration of sugar to-hydroxylevulinylaldehyde. This aldehyde condenses, in acidic medium, with diphenytumine to produce a deep-blue colored condensation products with absorption maximum at 595nm.
 
 



Materials

DNA Standard (0.5mg/mL)
Saline Citrate (0.15M NaCl, 0.015 M Na3 Citrate) Solution
Diphenylamine Reagent
    Mix 5g fresh or recrystallized diphenylamine, 500mL glacial acetic acid and 13.75mL cone. H2SO4. Stable for six months at 2°C; warm to room temperature and swirl to remix before use.
 
 
Procedure
1.
Prepare separate marked tubes containing 1mL, 2mL and 3mL aliquots of the isolated DNA dissolved in standard saline citrate and similar aliquots of a 0.5mg DNA/mL standard.
2.
Make all sample tubes, and a separate blank, up to 3mL with H2O. Add 6mL of diphenylamine reagent to each tube, and after mixing, heat the tubes in a boiling water bath for 10 min. Cool the tubes.
3.
Read the absorbance of blue solution at 600nm against the blank.
4.
Construct a standard graph A600 (ordinate) versus quantity of DNA (abscissa) and then calculate the concentration of DNA dissolved in the saline citrate solution.
5.
Prepare separate marked tubes containing 1mL, 2mL and 3mL aliquots of the isolated DNA dissolved in standard saline citrate and similar aliquots of a 0.5mg DNA/mL standard.
 
 
Notes
This method is commonly applied for samples of 50—500|Mg DNA. Burton has introduced a modified diphenylamine reagent containing acetaldehyde; in this reagent, samples are heated at 30°C for several hours for developing color.
 
 
References
1.  Burton, K (1956) BiochemJ 62 315.
2.  Ashwell, G (1957) In: Methods in Enzymol 3 (Eds Colowick, S P Kaplan, N O) Academic Press New York p 99.
3.  Sadasivam, S, Radhashanmugasundaram and Shanmugasundaram, E R B (1915) Arogya – J Health Sci 1 125.
 
     
 
 
     




     
 
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