商品编号


TSM08KR



品牌


Lucigen



公司


Lucigen



公司分类


In Vivo Tn5 Transposon Mutagenesis



Size

10 Reactions


商品信息

Generate random gene knockouts in non-
E. coli
bacteria


Generate mutants with improved genetics or function

Identify genes involved in pathogenesis, toxicity, biofilm development

Unravel metabolic pathways

Identify essential genes and regulatory elements

Characterize novel genes and gene functions

Insert Kan selectable
Marker
into gDNA and rescue clones in
E. coli
host expressing the
pir
gene product

100’s of citations for many different applications





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? ori
/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 (R6Kγ
ori
). 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 poss
IBL
e transformation efficiency. Activation of the Transposome by Mg
2+
in the cell results in the random insertion of the EZ-Tn5 ori
/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? ori
/KAN-2>Tnp Transposome? and TransforMax? EC100D?
pir
+
or TransforMax? EC100D?
pir
-116 Electrocompetent
E. coli.



*Covered by
issued and/or pending patents, exclusively licensed or assigned to Epicentre? (an
Illumina
? Company).

ORDER INFORMATION
EZ-Tn5? ori
/KAN-2>Tnp Transposome, KAN-2 FP-1 Forward Primer, R6KAN-2 RP-1 Reverse Primer, Sterile Water.