EZ-Tn5™ Protein Truncation Kit

EZ-Tn5™ Protein Truncation Kit


货号

EZI41110


规格

10 Rxns


存储温度

-20℃


产品详情


 

Applications

  • Generation of a library of N-terminal and C-terminal protein deletion clones for structural and functional studies.

The EZ-Tn5™ Protein Truncation Kit* provides a convenient method for generating a library of N-terminal and C-terminal protein deletion clones that can be expressed in E. coli. The kit features the EZ-Tn5 <p15Aori/KAN-2/T7Exp> Transposon, which is randomly inserted into any target DNA using a simple, in vitro reaction catalyzed by EZ-Tn5 Transposase. Then, the transposition reaction is amplified by PCR using one primer that anneals near a transposon end and another primer that anneals to a fixed point in the target sequence. Since the transposon is randomly inserted along the length of the target sequence, amplification generates a library of N-terminal or C-terminal deletions, depending on the choice of primers (Fig. 1).

Using the End-Repair Mix and Fast-Link DNA Ligase provided in the kit, the pool of PCR products is blunt-ended and self-ligated to create a library of kanamycin-resistant "rescue" clones that can replicate from the p15Aori in standard strains of E. coli. Rescue clones with an N-terminal deletion will also contain a transposon-derived T7-promoter region in a 5´→3´ orientation. At least one third of these clones will generate in-frame fusion proteins that can be expressed in cells containing a T7 RNA polymerase gene, e.g., E. coli BL21.

Benefits

  • Random transposon insertion ensures no bias in library construction.
  • N-terminal deletion clones contain a T7 promoter for expression studies.

*Covered by issued and/or pending patents.

Figure 1 (click to enlarge). The EZ-Tn5™ Protein Truncation Kit can be used to create random, unidirectional deletions from the 5´ end (shown here) or 3´ end of a target sequence.