商品编号


EZI011RK



品牌


Lucigen



公司


Lucigen



公司分类


In Vitro Tn5 Transposon Mutagenesis



Size

10 Reactions


商品信息

Random insertion of R6K origin for rescue of flanking DNA or plasmids


Insert R6K origin randomly into any DNA sequence
in vitro

Skip primer walking - simplify Sanger sequencing of large DNA inserts

Rescue clones in
E. coli
host expressing the
pir
gene product

Minimize insertion bias with the hyperactive Tn5 system, known for highest level of randomness






Applications

"Rescue" of plasmids or any other circularized DNA (e.g., mitochondrial DNA) that would not otherwise replicate in
E. coli
because they lack a recognizable origin of replication and/or a selectable
Marker
.

Preparation of DNA sequencing templates from transposon insertion clones without primer walking or additional subcloning.

Creation of a library of random gene knockouts
in vitro
to facilitate genetic analysis of plasmid-encoded genes.

The EZ-Tn5? ori
/KAN-2> Insertion Kit* facilitates the sequencing and genetic analysis of plasmids or any other circularized DNA that would not otherwise replicate in
E. coli.
1,2
The kit is based upon the EZ-Tn5? ori
/KAN-2> Transposon which carries the
E. coli
R6Kγ conditional origin of replication (R6Kγ
ori
) and a kanamycin resistance
Marker
. A simple, one-step, 2-hour
in vitro
reaction randomly inserts the transposon into the target DNA. Then an aliquot of the reaction is used to transform an
E. coli
host expressing the
pir
gene product, which is required for replication from the R6Kγ
ori.
Insertion clones are selected on kanamycin plates and can be sequenced bidirectionally using the provided primers that are homologous to the ends of the transposon. Clones can be maintained in Epicentre's TransforMax? EC100D?
pir
+
or TransforMax? EC100D?
pir
-116 strains.
3


Figure 1. A plasmid containing the EZ-Tn5? ori
/KAN-2> Transposon can be maintained in TransforMax? EC100D?
pir
+
cells at ~15 copies per cell (Lanes 1-4) or TransforMax? EC100D?
pir
-116 cells at ~250 copies per cell (Lanes 5-8).
Colonies from randomly chosen clones were processed using the Colony Fast-Screen? Kit. A 5-microliter aliquot of lysate was loaded per lane. M, supercoiled DNA ladder.

References

Jendrisak, J.
et al.
(2002)
Epicentre Forum

9
(1), 14.

Yoon, Y. and Koob, M. (2003)
Epicentre Forum

10
(2), 10.

Metcalf, W.W.
et al.
(1994)
Gene

138
, 1.


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

ORDER INFORMATION
EZ-Tn5? Transposase, EZ-Tn5? ori
/KAN-2> Transposon, EZ-Tn5? 10X Reaction Buffer, EZ-Tn5? 10X Stop Solution, Forward and Reverse Primers, Control Target DNA, Sterile Water.