Rapid and reliable cloning of PCR products.

Center for Biotechnology, University of Bergen, N-5020 Bergen, Norway The cloning of PCR products is often desirable, especially when characterizing heterogenous product populations. 1o achieve this, restriction sites are frequently included at the 5 ' end of each primer to allow direct cloning. However, low cloning frequencies are sometimes encountered, presumably due to incomplete digestion or Taq polymerase carryover./1,21 Adaptor and linker cloning strategies can be employed to circumvent these problems but standard protocols include several timeconsuming purification steps. Several members of our laboratory have experienced difficulty in obtaining PCR clones. The following protocol routinely generates high cloning efficiencies (106 transformants/ixg of vector) from previously recalcitrant PCR products. Self-ligation of linear PCR products generates multimeric DNA substrates for complete cleavage by restriction enzymes, t]) The concurrent incubation of Klenow, T4 polynucleotide kinase, and T4 DNA ligase employed here effectively creates concatemeric DNA substrates by polishing, phosphorylating, and ligating PCR termini in a single step (Fig. 1). A variation for linker and adaptor cloning requiring minimal manipulation is also shown. The linker protocol facilitates directional cloning when only a single restriction site is