Authors
Eric R. Geertsma and Bert Poolman
Summary
We have developed a generic method for high-throughput cloning in bacteria less amenable to conventional DNA manipulations. The method employs ligation-independent cloning in an intermediary Escherichia coli vector, which is rapidly converted via vector backbone exchange (VBEx) into a bona fide, organism-specific plasmid ready for high-efficiency transformation. Here, we describe the VBEx procedure for Lactococcus lactis. The procedure will yield L. lactis expression vectors from which the gene of interest can be expressed with a cleavable decaHis-tag or alternative tags at either the N- or C-terminus. Simultaneously, E. coli expression vectors holding similar tags can be created.Further details
The protocol was published on Protocol Exchange in 2007. To see the entire protocol, click on the source link.Advertisement
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