Among the advantages of transposon mutagenesis is that the transposon serves as a marker that can be used to clone and sequence the region of genomic DNA that has been disrupted. Nothing makes this cloning process easier than creating mutations in vivo with the EZ-Tn5™ <R6Kγori/KAN-2>Tnp Transposome™ Kit.* In addition to encoding a broad host-range kanamycin resistance gene, the transposon contains an E. coli conditional origin of replication (R6Kγori). The presence of this origin of replication enables the propagation or "rescue" of the region of genomic DNA, or plasmid, into which the transposon has been inserted.
First, electroporate the Transposome into electrocompetent cells of the highest possible transformation efficiency. Activation of the Transposome by Mg2+ in the cell results in the random insertion of the EZ-Tn5 <R6Kγori/KAN-2> Transposon into the host's genomic DNA. Select transposition clones on kanamycin plates or by screening for the loss of gene function.
|Figure 1 (click to enlarge). The process for rescue cloning of transposon insertion sites in genomic DNA using the EZ-Tn5™ <R6Kγori/KAN-2>Tnp Transposome™ and TransforMax™ EC100D™ pir+ or TransforMax™ EC100D™ pir-116 Electrocompetent E. coli.|