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Assignment

Molecular Weight Determination

The migration rate of DNA, RNA, and Protein through agarose and polyacrylamide gels increases non-linearly as the size of the macromolecule decreases. Estimation of the size of a macromolecule from it's migration, compared to the migration of standards of known size, is possible by plotting the size vs. the distance migrated. Since the curve is non-linear, many standard points are required to get an accurate estimate of an unknown molecule's size. An alternate method is to transform the data before plotting, such that the data estimates a linear response. Most molecular mobilities can be linearized by taking the log of the distance migrated and plotting it vs the size of the macromolecule. This can also be done by plotting the untransformed data on semi-log paper with the distance plotted on the log scale. Another useful transform in linearizing protein migration data is one that raises the size of the macromolecule to the (-2/3) power before plotting the transformed data against the distance migrated. The methods of linear regression, as monitored by R values (-1 or 1 = perfect linear fit), can determine the goodness of fit for each transform.

1- Using the standard size and MW data below, make a graph of the transformed standard data using the log and (-2/3) transforms. From the graphs, determine which method linearizes the data. Do a linear regression and find the slope (m) and y-intercept (b) and R value for the linear equation y=mx+b. Use the linear prediction equation to estimate the MW data for the unknown migrations.

2- Using the standard size and MW data below, make a graph of the transformed standard data using the log and (-2/3) transforms. From the graphs, determine which method linearizes the data. Do a linear regression and find the slope (m) and y-intercept (b) and R value for the linear equation y=mx+b. Use the linear prediction equation to estimate the MW data for the unknown migrations.