EZ::Tn5™ Transposomes™ the stable complex formed between an EZ::Tn5™ Transposon and EZ::Tn5™ Transposase™ provide a simple and reliable method for generating a library of random gene knockouts in vivo. Just electroporate the EZ::Tn5™ Transposome into any of a broad range of living cells and select for a marker encoded by the EZ::Tn5™ Transposon . Because there is no need for cell conjugation, suicide vectors, or specific host factors, EZ::Tn5™ Transposomes are ideal for creating mutants in species that have poorly described genetic systems or lack adequate molecular tools.

Ready-to-use EZ::Tn5™ Transposomes are available containing either a kanamycin selectable marker (<KAN-2>) or dihydrofolate reductase gene (<DHFR-1>) that can be selected on plates containing trimethoprim. These markers are readily expressed in many gram-negative bacteria. Or you can create your own EZ::Tn5™ Transposome using one of the EZ::Tn5™ pMOD™ Transposon Construction Vectors and EZ::Tn5™ Transposase.
All EZ::Tn5™ Transposons contain unique primer binding sites at each end for bidirectional sequencing. Hence, a gene knockout can be sequenced directly using bacterial genomic DNA as template and the primers provided with each Transposome. The transposon insertion can also be rescued and the flanking DNA sequenced when mutations are maded with an EZ::Tn5™ <R6Kγori /KAN-2>Tnp Transposome.

EZ::Tn5™ Transposome-mediated insertions have already been used successfully in a variety of bacteria including E.coli, Salmonella, Pseudomonas, Proteus, Mycobacterium, Moraxallas and others (Table 1). The number of transposition clones obtained is highly dependent on the transformation efficiency of the host cell. Electroporation of an E. coli strain with a transformation efficiency for pUC19 DNA of >10⁹ cfu/µg typically results in >10⁵ independent transposon insertion clones when 1µl of EZ::Tn5™ Transposome is used.

Number of Kan^R transposon insertion clones produced from electroporation of 1µl of EZ::Tn5™ <KAN> Tnp Transposome™

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

Applications

• Analyse gene function by creating gene knockouts (insertional mutations) in living cells without using suicide vectors, conjugation, or specific host factors
• Sequence bacterial genes without cloning using primers (provided in the kits) that are homologous to the ends of the inserted transposon
• Create mutations in vivo using an R6Kγori containing transposon and rescue clone the region of genomic DNA into which the transposon has been inserted
• Construct a custom EZ-Tn5™ Transposome using one of the EZ-Tn5™ pMOD Transposon Construction Vectors to introduce virtually any DNA sequence of interest into living cells

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