Procedure:

1. As the concentration of the unknown DNA sample can be anything, the assay will be performed with a range of dilutions (1, 1:10, 1:100, and 1:1000). Prepare 1 ml of each of the dilutions.
   
   
2. Take 15 test tubes and label them from 1 to 15.
   
   
3. Pipette out 100 μl , 200 μl , 300 μl , …….., 1000 μl calf thymus DNA standard in the glass tubes labeled 1 – 10; leave blank the tube no. 11.
   
   
4. Add distilled water to make the final volume 1 ml in each of the tubes (including blank).
   
   
5. Take 1 ml of each of the unknown DNA dilutions in the tubes labeled 12 – 15.
   
   
6. Add 3 ml of DPA reagent in each of the 15 tubes and mix well by vortexing.
   
   
7. Cover each of the tubes with the caps and place them in boiling water bath for 10 minutes.
   
   
8. Take out all the tubes from water bath and allow them to return to room temperature.
   
   
9. Measure the absorbance of tubes 1 – 10 and 12 – 15 at 595 nm in the quartz/glass cuvette against the reagent blank (tube 11).
   
   
10. Record the readings in the suggested observation table below:

Observation Table:

Table 6.1: Observation table for the diphenylamine assay