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. |
