Generation of Random Fragments by Sonication

Except for all but the shortest of sequencing projects, the resolution limits of polyacrylamide gels (being somewhat less than 1 kb) prevents determining the entire sequence in one run. There are several ways to overcome this by subcloning or by primer walking. Each has its advantages and disadvantages. These alternatives should be considered whenever a new project is undertaken. Frequently, the best approach would in fact use more than one method. A high proportion of large-scale sequencing projects use random cloning and sequencing (shotgun sequencing) to produce in excess of 95% of the data, and then employ more directed means to complete it (1 –3 ).