Epicentre® offers a variety of easy-to-use EZ-Tn5™ Transposon Tools that will make almost any sequencing project faster and easier using one of 3 basic strategies:

1. The In Vitro Insertion Strategy

The In Vitro Insertion Strategy will simplify and speed up your sequencing project if you already have cDNA or genomic clones that are too big to sequence with a single set of sequencing reactions. You don't need experience with transposons to use this strategy. It's easy! Just incubate your clone for 2 hours with a KanR, TetR or TpR EZ-Tn5™ Transposon and EZ-Tn5™ Transposase provided in an EZ-Tn5™ Insertion Kit, transform competent E. coli. with an aliquot of the reaction mix, and select insertion clones on medium containing the transposon-encoded antibiotic. You will obtain millions of different insertion clones per reaction - enough to sequence even the largest BAC clone. And the complete sequence is obtained using the single set of bi-directional transposon-specific sequencing primers provided in the kit.

2. The Transposome™ Strategy

The Transposome Strategy is useful if you're interested in finding a gene related to a phenotype. An EZ-Tn5™ Transposome is a stable complex formed between the EZ-Tn5™ Transposase and EZ-Tn5™ Transposon in the absence of Mg2+. An EZ-Tn5™ Transposome is so stable that it can be electroporated into living cells (e.g., E. coli, Proteus vulgaris, S. typhimurium, Pseudomonas sp., Rhodococcus sp., and Mycobacterium sp.), where it is activated by intracellular Mg2+ and inserts a single transposon randomly into the host's genomic DNA. A library of mutants, each with an independent random transposon insertion, can be selected on antibiotic medium. In many cases, phenotypic changes caused by a gene 'knockout' can be identified. Then the insertion site in the gene can often be sequenced directly without cloning using total bacterial genomic DNA and provided primers that are homologous to the ends of the inserted EZ-Tn5™ Transposon. An EZ-Tn5™ Transposome that contains an R6Kg origin of replication, enabling easy rescue cloning of DNA flanking the transposon insertion site, is also available.

3. The In Vitro Deletion Strategy

The In Vitro Deletion Strategy should be considered if you don?t already have clones and need to generate a new plasmid or cosmid library for a sequencing project, or need to subclone a large DNA for sequencing or functional analysis. To use this strategy, clone your DNA into the multiple cloning site of one of the specially-constructed plasmid or cosmid deletion vectors and transform E. coli. Random unidirectional deletions can be generated in any of the individual clones simply by incubating the clone with EZ-Tn5™ Transposase. After transforming E. coli, up to about 35% of the colonies will contain random deletion subclones, which can be identified on antibiotic plates and sised on a gel. The complete sequence of each original primary clone can be obtained by choosing a set of random deletion subclones spanning the entire size range and sequencing using the provided primers, which are homologous to a site on the vector adjacent to the start of the deletion.

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