FIGURE 6.4 - Diagram of BACs digested with NotI. Lanes 3 and 42 contain the PFGE lambda ladder. All of the sample lanes possess vector DNA except those demarcated by white rectangles. The absence of DNA in these lanes indicates that the BAC DNA was lost during the miniprep protocol. Though most lanes contain one "non-vector" band, some lanes contain several non-vector bands (lanes marked with light blue rectangles). The presence of multiple bands indicates that the insert in question contains one or more NotI restriction sites. The insert size for a particular clone is the sum of the base pair lengths of its non-vector bands. For example, in lane 5, the sole insert fragment has a mean length of approximately 130 kb. In lane 8, the insert size is (50 kb + 70 kb =) 120 kb. If the transformation procedure was successful, there should be few (if any) "false positives" (white clones containing a vector but not a piece of exogenous insert DNA. In the figure, examples of false positives are enclosed in red rectangles. Each false positive has a vector band but no insert band(s).

In general, average insert length is best determined using an image capture system with software capable of determining DNA lengths based on a comparison of sample bands to those of a ladder.