Fig. 3 Examples for each step are from the creation of various libraries. (a) Example for a primer pair,
important sequence features are highlighted. (b) Examples for PCR products ranked from suitable to
unsuitable for subsequent steps. Troubleshooting recommendations are indicated. Start a library with the
least efficient PCR, because the required number of transformants increases while performance usually
declines in successive rounds of mutagenesis. (c) Efficiency of the ligation should be tested before
electroporation. A circular vector will give a single band (1) when digested with a single cutting restriction
enzyme, if the ligation is incomplete, it will result in two extra bands with different intensities depending on the
ligation efficiency (2, 3). No ligation results in just a double band (4). Reason for incomplete ligation can be the
presence of side products capping the linear DNA (compare PCRs labeled # and * in b). Transformation of 3 or
4 did not yield any or sufficient numbers of colonies, while transformation of 2 was still sufficient
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Martin Spinck et al.