The In Vitro Insertion Strategy is used to randomly insert an EZ-Tn5™ Transposon, containing a selectable marker and sequencing primer binding sites, into cloned DNA in a simple 1-step, 2-hour enzymatic reaction.
The hyperactive EZ-Tn5™ Transposon system permits one to obtain transposon insertions even in large or low-copy number plasmid, cosmid, fosmid and BAC clones. Target DNA does not need to be CsCl purified. In vitro reaction conditions have been optimised to maximise transposon insertion efficiency while minimizing multiple insertion events. In a typical in vitro reaction, the transposon insertion efficiency is 1-10% yielding up to >10⁶ transposon insertion clones - each containing a single randomly inserted EZ-Tn5™ Transposon. Because the insertion process is highly random, the result is a complete population of insertion clones covering the target DNA.

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

Applications

• All EZ-Tn5™ Transposons can be used to facilitate complete sequencing of plasmid, cosmid, fosmid and BAC clones faster and easier than by primer walking or subcloning
• Use the EZ-Tn5™ In-Frame Linker Insertion Kit to make random 57-nucleotide (19-amino acid) in-frame insertions into genes of expressed proteins for protein engineering, functional analysis, and domain or epitope mapping
• Rapidly characterise plasmids that otherwise would not replicate in E. coli by randomly inserting an E. coli conditional origin of replication (R6Kγori) and a kanamycin resistance marker using the EZ-Tn5™ <R6Kγori /KAN-2> Insertion Kit
• Introduce a phage T7 promoter into cloned DNA using the EZ-Tn5™ <T7 /KAN-2> Insertion Kit and generate RNA transcripts in vitro or in vivo from the insertion site
• Insert any DNA sequence of interest (e.g., selectable marker, reporter gene, control element) into target DNA in vitro by making a custom EZ-Tn5™ Transposon with one of the EZ-Tn5™ pMOD™ Transposon Construction Vectors

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