EZ-Tn5™ In-Frame Linker Insertion Kit

EZ-Tn5™ Transposase

TransforMax™ EC100™ Electrocompetent E. coli

*TransforMax™ EC100™ Electrocompetent Cells are recommended for high-efficiency, non-size-biased cloning of DNA containing EZ-Tn5™ Transposon insertions.

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

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Last modified: 2008-07-15 14:57:15

Make random, 57-bp (19-amino acid) in-frame insertions into genes of expressed proteins for protein modification, functional analysis, and domain or epitope mapping.

The EZ-Tn5™ In-Frame Linker Insertion Kit is a transposon-based protein modification system that was designed to rapidly and easily produce random 57-basepair (19 amino acid) insertions into a cloned DNA (e.g., genes and cDNA) that encodes an expressed protein. Since the inserted 19 codons are designed to be readable in all three reading frames, the kit can be used to identify key regulatory, binding, catalytic, and permissive and non-permissive sites in proteins.

The kit features the EZ-Tn5™ <Not I /KAN-3> Transposon, which contains a kanamycin-resistance (Kan^R) marker flanked by Not I restriction sites (Figure 1). A simple in vitro enzymatic reaction randomly inserts a single EZ-Tn5 <Not I /KAN-3> Transposon into each clone and produces >10⁶ kanamycin-resistant insertion clones.
Insertion clones for further analysis can be identified by gene functional analysis or by mapping or DNA sequencing of the transposon insertion site. Once clones are chosen, the kanamycin resistance gene is excised from the transposon by Not I digestion (Figure 2). Each Not I-digested clone is then ligated and transformed into high-efficiency E. coli (e.g., TransforMax™ EC100™ Electrocompetent E. coli). The resulting clones each contain a single, random 19-codon insertion that can be read to express protein in all three reading frames. Thus, the protein retains its original amino acid sequence on both sides of the insertion site.

Two un;abelled primers are provided with the kit to facilitate sequencing or mapping of the EZ-Tn5™ Transposon Insertion site.

Figure 1. The EZ-Tn5™ In-Frame Linker Insertion Kit is based on the highly random Tn5 transposition system. A single in vitro reaction generates thousands of insertion clones-each containing a different transposon insertion.

Figure 2. The EZ-Tn5™ <Not I/KAN-3> Transposon contains a kanamycin resistance gene flanked by Not I restriction sites. A 19-codon insertion that can be read in all three reading frames is generated following Not I digestion and ligation.

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

Applications

• Make random 57-basepair (19 amino acid) insertions that are readable in all three reading frames in any cloned DNA that encodes an expressed protein for protein domain mapping, protein engineering, or epitope mapping
• Simplify and speed up complete sequencing of cDNA and genomic clones
• Modify important DNA sequences such as promoter regions or binding motifs

Benefits

• Efficient - generate a population of >10⁶ insertion clones, each with a single randomly inserted transposon, to provide complete coverage of the clone
• More versatile than traditional linker scanning mutagenesis - the EZ-Tn5™ Transposon insertion is random and not limited to pre-existing restriction endonuclease sites in the cloned DNA
• Unique Not I restriction site in the linker insertion facilitates mapping and additional gene construction

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

Protocols for: EZ-Tn5™ In-Frame Insertion Kit

(catalogue number EZI04KN)

(catalogue number KN0801 / NKFP072 / NKRP062)

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

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

References

1. Hoffman, L.M. and Loomis, K.B. (2000) EPICENTRE Forum 7 (3), 4.
2. Bailey, J. and Manoil, C. (2001) EPICENTRE Forum 8 (1), 1.

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