An improved method for estimation of DNA fragment lengths from mobility on agarose gels is introduced. Commonly, this estimation is done by graphing the logarithm of length against the mobility of standards, although this procedure produces a noticeably curved plot. The method presented here is based on the relationship of E. M. Southern (1979, Anal. Biochem., 100, 319–323), who showed that the reciprocal of mobility plotted against fragment length is linear. A least-squares analysis has been developed to use this relationship together with any number of standards to estimate fragment length. The method also tests internal consistency of the assigned lengths of a set of fragments. This analysis has been applied to sets of restriction fragments of λ DNA, indicating errors or inconsistencies in previously assigned lengths. Using a more consistent set of λ values, the lengths of pBR322 fragments of known sequence were predicted with better than 1% accuracy. No evidence of sequence-dependent migration was seen. A computer program to do this analysis is included.
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