Quantification of DNA in unknown sample by diphenylamine (DPA) reagent.

Chemicals

1. Standard DNA solution (0.25mg/ml),
2. Diphenylamine reagent
3. DNA sample in saline citrate buffer
4. Saline citrate buffer (0.15M NaCl, 0.015M sodium citrate, pH 7.0)
5. Glacial acetic Acid
6. Concentrated H₂SO₄

Glassware/Plastic ware

All glassware and plastic ware used should be sterilized:

1. Test tubes
2. Micro pipettes
3. Graduated Cylinder

Instruments/ equipment

1. Spectrophotometer
2. Water bath

Preparation of diphenylamine reagent

1. Dissolve 1 g of diphenylamine in 100ml of glacial acetic acid.
2. In this, now add 2.5 ml sulphuric acid.

When DNA is treated with diphenylamine under the acidic condition a blue colored complex is formed which has an absorption peak at 595nm.This reaction is given by 2- deoxypentose in general. In acidic solution deoxypentose are converted into a highly reactive β hydroxyl leavulinic aldehyde which reacts with diphenylamine gives blue complex. In DNA, only the deoxyribose of purine nucleotide reacts so that the value obtained represents one half of the total deoxyribose produced.

Reactions-

1. Pipette out 0.2, 0.4, 0.6, 0.8 and 1 ml of working standard in to the series of labelled test tubes.
2. The final volume in all the test tubes was made to 1 ml with distilled water.
3. Pipette out 1 ml of the given sample in another test tube which serves as a blank.
4. Pipette out 0.5ml of unknown DNA sample in another test tube labelled as 'unknown' and volume was made upto 1ml with distilled water.
5. Now add 5 ml of DPA reagent to all the test tubes including the test tubes labelled 'blank' and 'unknown'.
6. Place all the test tubes in a boiling water bath for 15 minutes.
7. Now, cool the test tubes and note down the absorbance of the blue colored solution at 595 nm against blank.
8. Plot the standard curve by taking concentration of DNA in βg /ml along X-axis and absorbance at 595 nm along Y-axis.
9. From this standard curve calculate the concentration of DNA in the unknown sample.

straight line graph is formed by taking concentration of standard DNA in ug/ml along Xaxis and absorbance at 595nm along the Y-axis.

Calculation

Slope = y2-y1/x2-x1

The amount of the DNA present in the given unknown solution = ________μg/ml.

Deoxyribose in presence of acid converts to a compound that binds with diphenylamine to form a blue coloured complex. In standard DNA solution the gradient of blue colour represents the corresponding DNA concentration.
Optical density of lowest concentrated DNA (100 βg/ml) is _____ and optical density of highest concentrated DNA (1000 µg/ml) is______. The concentration of unknown DNA is ___ βg/ml.

1. It is recommended to use freshly prepared diphenylamine reagent. The solution however, can be stored in advance in dark at 2-8 ⁰C
2. 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.
3. Clean all the test tubes properly before use.
4. Spectrophotometer should be switched on 15-30 minutes before use to allow it to warm up.

1. Estimation of DNA (1)
2. Estimation of DNA (2)
3. Estimation of DNA (3)