ANDC DU/Biology Protocols/DNA Quantification
To perform the quantitative estimation of DNA using UV spectrophotometer and to determine the absorption maxima.
Principle
Absorption spectroscopy has long been the method of choice to measure the amount of DNA or RNA in a solution. Purines and pyrimidines in nucleic acids absorb UV light because of presence of conjugated double bonds of the constituent purines and pyrimidines bases. These have absorbance maxima at wavelengths of 260 nm which is linearly correlated with the concentration of DNA or RNA in the solution For double stranded DNA , one A260 or OD260 unit corresponds to concentration of 50ug/ml and for single stranded DNA and RNA its 40 ug/ml (for double stranded DNA, the average extinction coefficient is 50(ug/l)-1cm-1 for single stranded DNA or RNA the value is 40(ug/l)-1cm-1 The Lambert Beers law is valid at least to an OD=2 and the concentration of solution of nucleic acid is therefore easily calculated by simple interpolation.
Requirements
Biological
DNA sample
Chemicals
TE buffer
Instrument/Equipment
UV spectrophotometer Micropipettes
Glass ware/Plastic ware
Eppendroffs Microtips
Procedure
A. Quantification of DNA
• Make two or three dilutions of the DNA samples • Switch ON the spectrophotometer and adjust the wavelength at 260nm. • Set the instrument at zero absorbance with TE buffer. • Take the OD of the samples. • Calculate the concentration of DNA in the samples from the following formulae
Concentration of double stranded DNA in solution (ug/ml) = 50 x A260 x dilution factor.
• Take the mean of the various dilutions as the concentration of the DNA.
B. Estimation of Absorption Maxima.
• Take a known amount of DNA solution. • Switch ON the spectrophotometer and adjust the wavelength at 200nm. • Set the instrument at zero absorbance with TE buffer. • Take OD of the sample. • Take the readings at various wavelengths such as 230nm, 260nm, 300nm and 320nm. • Plot a graph between Wavelength and OD.
Result
Discussion
Precautions
• Ethidium bromide should be handled carefully with gloved hands. • Do not touch the gel when electric current is ON. • Do not see UV radiations with naked eyes.
