Description
The End-Repair Mix converts DNA containing damaged or incompatible 5′- and/or 3′-protruding ends to 5′-phosphorylated, blunt-ended DNA. The conversion to blunt-ended DNA is accomplished by exploiting the 5′→3′ polymerase and 3′→5′ exonuclease activities of T4 DNA Polymerase (P7080). T4 Polynucleotide Kinase (Y9040) ensures that the ends of the blunt-ended DNA fragments are 5′-phosphorylated for subsequent ligation by T4 DNA Ligase Ligation may be performed immediately using Rapid T4 DNA Ligase (L6030-HC). The low concentration End-Repair Mix formulation is optimized for procedures such as general cloning using low DNA concentrations. The high concentration End-Repair Mix formulation is optimized for procedures such as library construction for next-generation sequencing using high DNA concentrations. ATP is not required because the T4 Polynucleotide Kinase can utilize the deoxynucleotides (dATP and dTTP) used in the blunting reaction mix as phosphate donors. The enzyme mixes are supplied in 100 mM KCl, 10 mM Tris-HCl, 0.1 mM EDTA, 1 mM DTT, 0.1% Triton X-100, 50% glycerol; pH 7.4 at 25°C. The enzyme mixes are supplied with 1 mM dNTPs (N2060) and 10X End-Repair Buffer (B9140) containing 1 M Tris-HCl, 500 mM NaCl, 100 mM MgCl 2, 50 mM DTT, 0.25% Triton X-100; pH 7.5 at 25°C.
Test Specification Purity >99% 3’→5’ nuclease Functional 5’ phosphorylation Functional 5’→3’ DNA synthesis Functional Double-stranded endonuclease 10 µL = No conversion E. coli DNA contamination 10 µL <10 copies
The End-Repair Mix produces blunt-ended 5′-phosphorylated DNA by repairing DNA with damaged or incompatible 5′-protruding and/or 3′-protruding ends. The conversion to blunt-ended DNA is achieved by exploiting the 5′→3′ polymerase and 3′→5′ exonuclease activities of T4 DNA Polymerase (P7080). The action of T4 Polynucleotide Kinase (Y9040) in the mix ensures that the ends of the blunt-ended DNA fragments are 5′-phosphorylated for subsequent ligation. The end-repaired DNA can be efficiently and rapidly blunt-end ligated into plasmid, cosmid, fosmid, BAC, other cloning vectors, or next-generation DNA sequencing adaptors.
Instructions for using End-Repair Mix (high and low concentration) are provided in the corresponding kit protocol in the resources below. Quality Control The purified enzymes in the End-Repair Mix (T4 DNA Polymerase P7080L and T4 Polynucleotide Kinase Y9040L) are free of contaminating endonucleases. In addition, >99% enzyme purity was analyzed by SDS-PAGE. Negligible E. coli genomic DNA was confirmed by qPCR. In a functional assay, 2 μL of End-Repair Mix was added to a double restriction enzyme digested, dephosphorylated plasmid DNA in 1X reaction buffer containing 0.1 mM dNTPs and incubated at 25°C for 30 minutes. Competent cells were transformed with the ligation mixture, plated onto LB/Amp/X-Gal plates and incubated overnight at 37°C. Control reactions consisting of End-Repair Mix without T4 DNA Polymerase and/or T4 Polynucleotide Kinase were tested in parallel. The efficiency of the reaction was evaluated by comparing the number of blue and white colonies present in the End-Repair Mix plates to those of the control plates.
