End-Repair Mix (LC) (200 rxn)

This enzyme formulation from QIAGEN converts DNA containing damaged or incompatible 5′- and/or 3′-protruding ends into 5′-phosphorylated, blunt-ended DNA. The conversion exploits the 5′→3′ polymerase and 3′→5′ exonuclease activities of T4 DNA Polymerase, while T4 Polynucleotide Kinase ensures that the blunt ends are 5′-phosphorylated for subsequent ligation. ATP is not required because the kinase can utilize the dATP and dTTP supplied in the blunting reaction mix as phosphate donors.

The low-concentration formulation is optimized for general cloning and other procedures using low DNA amounts, while the high-concentration version is intended for library construction for next-generation sequencing with higher DNA inputs. End-repaired DNA can be blunt-end ligated into plasmid, cosmid, fosmid, BAC, other cloning vectors, or next-generation DNA sequencing adaptors, and ligation may be performed immediately using a Rapid T4 DNA Ligase.

Assay specifications report >99% purity by SDS-PAGE, with 3′→5′ nuclease, 5′ phosphorylation, and 5′→3′ DNA synthesis all confirmed as functional. Double-stranded endonuclease activity is undetectable at 10 µL, and E. coli DNA contamination is <10 copies per 10 µL. The enzyme mix is supplied in 10 mM Tris-HCl, 100 mM KCl, 1 mM DTT, 0.1 mM EDTA, <0.1% Triton X-100, 50% glycerol (pH 7.4 at 25°C), together with 10X End-Repair Buffer and 1 mM dNTPs. The recommended protocol is incubation at room temperature (25°C) for 30 minutes, followed by heat inactivation at 75°C for 20 minutes. The product is intended for in vitro use only.