![]() Experiments made use of a common reporter vector and a set of modular primers to clone DNA fragments of increasing size. Overall, PIPE achieved cloning efficiencies of ∼95% with few manipulations, whereas SLIC provided a much higher number of transformants, but required additional steps. Our data suggest that for small inserts (<1.5 kb), OEC is a good option, requiring only two new primers, but performs poorly for larger inserts. These ligation-independent cloning approaches constitute an essential part of the researcher's molecular-tool kit. Principles of polymerase incomplete primer extension (PIPE) cloning, sequence and ligation-independent cloning (SLIC) and overlap extension cloning (OEC). (A) In PIPE, incomplete extension during PCR generates 3′-recessed ends. In SLIC, purified PCR products are treated with T4 DNA polymerase (DNAP) so that the exonuclease activity will increase the proportion of recessed ends. In both these techniques, by amplifying vector and insert with primers containing complementary 5′-tails and mixing the products, the overhangs can anneal and are joined in vivo after transformation into E. In OEC, the insert PCR product acts a megaprimer to generate a nicked plasmid by overlap extension in vitro in a second round of amplification. For all techniques, a DpnI digestion step is included to remove plasmid template (not shown).
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