Calculations:

 

1. Plot the absorbance values obtained for tubes 1 – 10 against the amount of standard DNA added to these tubes.
   
   
2. Fit the data points using linear regression [with intercept (0,0)].
   
   
3. Determine the slope of the regression line.
   
   
4. The concentration of the unknown DNA is given by:
   
   

Notes:

1. It is recommended to use freshly prepared diphenylamine reagent. The solution, however, can be prepared in advance and stored in dark at 2 – 8°C.
   
   
2. Prepare all the samples in triplicate and the tubes should be labeled properly. To follow the numbering used in the procedure and table given above, the tubes can be labeled as a, b, and c ; for example the three samples for tube 1 can be labeled as 1a, 1b, and 1c. The calculations can then be performed taking the average absorbance of the three tubes.
   
   
3. Concentrated sulfuric acid should be carefully pipetted out using a 5 ml glass pipette with the help of a pipette aid in a chemical fume hood.
   
   
4. The dilution(s) of unknown sample that show absorbance between 0.05 – 1.0 should be used for calculations.
   
   
5. UV/Visible spectrophotometer should be switched on at least 30 min before use.