End-Repair Mix

• Utilizes the 5′→3′ polymerase and 3′→5′ exonuclease activities of T4 DNA Polymerase
• Includes T4 Polynucleotide Kinase to 5′-phosphorylate the ends of the blunt-ended DNA
• Low-concentration formulation prepares ≤1 µg of DNA for blunt-end ligation
• High-concentration formulation prepares >1 µg of DNA for blunt-end ligation
• Blunted DNA is suitable immediately for ligation by T4 DNA Ligase

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 MgCl₂, 50 mM DTT, 0.25% Triton X-100; pH 7.5 at 25°C.

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 P7040 and T4 Polynucleotide Kinase Y9040) 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.

These products are available for molecular biology applications such as:

• Preparation of sheared or restriction enzyme digested genomic DNA for ligation of next-generation DNA sequencing adaptors
• Preparation of double-stranded cDNA, produced from cellular RNA transcripts, for ligation of next-generation DNA sequencing adaptors
• Preparation of sheared or digested DNA for blunt-end ligation into plasmid, cosmid, fosmid, or BAC vectors
• Preparation of DNA amplified by PCR, containing A-overhangs, for efficient and cost-effective blunt-end cloning