Quantitative Analysis of DNA
Aim: To determine the amount, concentration and purity of the given DNA sample
Principle: This experiment is purely an application of the Beer Lamberts’ Law which states that the concentration of the sample is directly proportional to the absorbance of light done by the sample. It is given by the following expression
The device UV spectrophotometer works on this principle and used to find the concentration of the sample.
Concentration and quality of a sample of DNA are measured with a UV spectrophotometer.
A standard graph can be drawn using different concentrations of DNA and OD (optical density) values.
The diagram above shows that a beam of monochromatic radiation (Io) is directed to a sample solution. Absorption takes place by the sample and the beam of radiation is leaving out (I)
- DNA sample
- TE buffer
- UV spectrophotometer
- Take the DNA sample (10 ul) in TE buffer
- Now dilute the above sample by the factor of 100 i. e, by taking 10µl of the sample in 990µl of TE buffer.
- After doing this take the optical density value at A260 & A280 and calculate the amount of DNA recovered
- Use the following formula to determine the concentration of DNA:
Total DNA (ug) = (A260) (50 ug/ml/A260) (100) (0.1 ml)
- where 100 is the dilution factor and 0.1 ml is the total volume of the DNA;
DNA quality measurement is based on the fact that OD at 260 nm is twice that at 280 nm if the solution contains pure DNA. If there is a contaminant, there is some additional OD, which decreases the OD ratio between 260 and 280 nm
Clean DNA has an OD260/OD280 between 1.8 and 2.0