Determination of Molecular Weight

This is done using an agarose gel electrophoresis of known molecular weight nucleic acids (ladder) along with the protein or nucleic acid to be characterised. A linear relationship exists between the logarithm of the molecular weight of native nucleic acid, and its Rf. The Rf is calculated as the ratio of the distance migrated by the molecule to that migrated by a marker dye-front.

A simple way of determining relative molecular weight by electrophoresis is to plot a standard curve of Rf vs. logMw for known samples (ladder), and read off the logMw of the sample after measuring distance migrated on the same gel. After introducing the data of ladder and sample Rf, sample Mw is calculated.

A line in a two dimensional or two-variable space is defined by the equation Y=ax+b , in full text: the Y variable can be expressed in terms of a constant (b) and a slope (a) times the X variable. The constant is also referred to as the intercept, and the slope as the regression coefficient. The correlation coefficient (r) is a quantity that gives the quality of a least squares fitting to the original data and is calculated as:

R ={Sum(x*y) - Sum(x)*Sum(y)/N} / sqrt {Sum(x**2) - Sum(x)**2/N}*{Sum( y**2) - Sum(y)**2/N}

Linear relationship between logarithm of the molecular weight and Rf :

logMw = aRf + b

is calculated as:

a = {Sum(x*y) - Sum(x)*Sum(y)/N} / {Sum(x**2) - Sum(x)**2/N}
b = Sum(y)/N - a*Sum(x)/N