Three basic principles form the basis for RNA-DNA measurements. These are:
Specific ratios determined from the above information can be used to determine the purity and type of nucleic acid present in a sample. Warbug and Christian(1) used these fundamental facts to determine how to quantify nucleic acids and proteins.
Click on the calculation of interest to you:
This equation is used to determine if extracted nucleic acid samples that have been purified are free from contamination by other molecules such as proteins and chemicals like phenol. The absorbance of the sample at 260 nm and 280 nm is measured. For pure DNA, the 260/280 is 1.8 and for pure RNA the 260/280 is 2.0. Values significantly lower than these indicate contamination of the sample.
The system uses the equation:Ratio = (A[260.000] - Bg[320.000])/(A[280.000] - Bg[320.000])
Warburg and Christian calculated a way to relate absorbance to specific factors and equations to give a concentration in µg/mL. Warburg and Christian determined these equations and factors based on the relationship between the molar absorptivity values of a yeast RNA and protein called enolase, determined at 260 and 280 nm with background correction at 320 nm. The factors they established are standard values and can be found in text books such as Manniatis.
They found that at 260 nm, an absorbance of 1.0 is equivalent to:
From these values, quantities can be calculated for other absorbances. For example, if a DNA preparation has an absorbance of 0.345 at 260 nm, the concentration of DNA will be 17.25 µg/mL (0.345 * 50 = 17.25).
Similarly, proteins also have set concentrations for absorbances at particular wavelengths.
The system uses the following equations to calculate concentrations:
[Protein] (A2 (280) * Protein Factor 2) - (A1 (260) * Protein Factor 1),
[Nucleic Acid] (A1 (260) * Nucleic Factor 1) + (A2 (280) * Nucleic Factor 2)
The RNA-DNA application can also calculate the absorbance at 260 nm multiplied by a factor.
The system uses the equation:
A1 * F1 [A1 (260) * Protein Factor 1]
(1) Warburg, O. and Christian, W. (1942) Biochem Z., 310, 384
RNA-DNA How to
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