End-Repair Mix (HC) (75 rxn)

A high-concentration enzyme formulation from QIAGEN, this End-Repair Mix converts DNA containing damaged or incompatible 5′- and/or 3′- protruding ends into 5′-phosphorylated, blunt-ended DNA suitable for blunt-end ligation. The conversion is accomplished by exploiting 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 by T4 DNA Ligase. The formulation is compatible with applications requiring >1 microgram of DNA.

Functionality is verified by adding 2 µL of End-Repair Mix to a double restriction enzyme-digested, dephosphorylated plasmid DNA in 1X reaction buffer containing 0.1 mM dNTPs and incubating at 25 °C for 30 minutes; competent cells transformed with the ligation mixture are plated onto LB/Amp/X-Gal plates and incubated overnight at 37 °C. The preparation is purified free of contaminating endonucleases, exhibits >99% enzyme purity as determined by SDS-PAGE, and contains <10 copies of E. coli genomic DNA as confirmed by qPCR.

The mix is supplied in 10 mM Tris-HCl, 100 mM KCl, 1 mM DTT, 0.1 mM EDTA, <0.1% Triton X-100, and 50% glycerol at pH 7.4 @ 25 °C, and is accompanied by 10X End-Repair Buffer (1 M Tris-HCl, 500 mM NaCl, 100 mM MgCl2, 50 mM DTT, 0.25% Triton X-100, pH 7.5 @ 25 °C). A typical reaction contains 1–19 µL of purified DNA (up to 5 µg), 2.5 µL of 10X End-Repair Buffer, 2.5 µL of 1 mM dNTP mix, and 1–3 µL of End-Repair Enzyme Mix, made up to 25 µL total with sterile water; after a 30-minute incubation at room temperature (25 °C), the enzyme is heat-inactivated at 75 °C for 20 minutes. Storage temperature is -25 °C to -15 °C. The product is for in vitro use only.