Transposomics™ Research |
In Vivo Transposomics™ Research
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Applications
- Rescue cloning of transposon-mutagenized microbial genes (Fig. 1).
- Rescue of plasmids from non-E. coli bacteria.
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.
Benefits
- Easily recover and propagate plasmids from diverse bacterial genera that
will not normally replicate in E. coli.
- Simple rescue cloning process of mutagenized genes speeds up structure/function
studies and sequencing.
- Random insertion of transposon DNA assures excellent coverage of entire
bacterial chromosome.
- Rescue clones can be sequenced bidirectionally using the primers provided
that are homologous to the ends of the transposon.
|
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. |
*The use of Transposome™ complexes for in vivo insertion of
a transposon, including, but not limited, to HyperMu™ and EZ-Tn5™ Transposome™ complexes,
is covered by U.S. Patent No. 6,159,736 and related patents and patent applications,
exclusively licensed to EPICENTRE.
End-It™ DNA End-Repair Kit
EZ-Tn5™ Transposon Construction Vectors
Fast-Link™ DNA Ligation Kit
TransforMax™ EC100D™ pir+ and pir-116 Electrocompetent E. coli
TypeOne™ Restriction Inhibitor
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