EZ-Tn5™ <R6Kγori /KAN-2> Insertion Kit

EZ-Tn5™ Transposase

Sterile Nuclease-Free Water

For Research Use Only. EZ-Tn5™ products covered by issued and pending patents.

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Last modified: 2008-07-24 11:19:45

The EZ-Tn5™ <R6Kγori /KAN-2> Insertion Kit facilitates sequencing and genetic analysis of plasmids or any other circularised DNA that would not otherwise replicate in E. coli. The kit features the EZ-Tn5™ <R6Kγori /KAN-2> Transposon which carries the E. coli R6Kγ conditional origin of replication and a kanamycin resistance marker
. A simple, one-step 2 hour in vitro reaction is performed to randomly insert the EZ-Tn5™ <R6Kγori /KAN-2> Transposon into your target DNA. Use an aliquot of the reaction to transform an E. coli host expressing the pir gene product, which is required for replication from the R6Kγori. 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

Epicentre® offers two strains of electrocompetent E. coli which express the pir gene product (Figure 1). TransforMax™ EC100D™ pir⁺ cells can maintain R6Kγ ori - containing plasmids at approximately 15 copies per cell, for cloning of potentially toxic or unstable sequences. TransforMax™ EC100D™ pir-116 cells are for high copy propagation of up to 250 plasmid copies per cell.

Figure 1. A plasmid containing the EZ-Tn5™ <R6Kγori/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-µl aliquot of lysate was loaded per lane. M, supercoiled DNA ladder.

Last modified: 2008-05-14 16:46:46

Applications

• 'Rescue' plasmids or any other circularised DNA that would not otherwise replicate in E. coli because they lack a recognisable origin of replication and/or a selectable marker
• Prepare DNA sequencing templates from transposon insertion clones and completely sequence the clone without primer walking or additional subcloning
• Create a library of random gene knockouts in vitro to facilitate genetic analysis of plasmid-encoded genes

Last modified: 2008-05-14 16:46:46

Protocols for: EZ-Tn5™ <R6Kγori/KAN-2> Insertion Kit

(catalogue number EZI011RK)

(catalogue number R6K0901 / KFP9122 / R6KRP091)

Please note: all protocols off site are the responsibility of the products supplier

Last modified: 2008-05-14 16:46:46