End-Repair Mix

• Converts DNA containing damaged or incompatible 5′- and/or 3′-protruding ends to 5′-phosphorylated, blunt-ended DNA
• High-concentration formulation optimized for high DNA concentrations, such as library construction for sequencing
• Low-concentration formulation optimized for low DNA concentrations, such as general cloning

The End-Repair Mix converts DNA containing damaged or incompatible 5ʹ- and/or 3ʹ-protruding ends to 5ʹ-phosphorylated, blunt-ended DNA. The conversion to blunt-ended DNA is accomplished by exploiting the 5ʹ→3ʹ polymerase and 3ʹ→5ʹ exonuclease activities of T4 DNA Polymerase (cat. no. P7080). T4 Polynucleotide Kinase (cat. no Y9040) ensures that the ends of the blunt-ended DNA fragments are 5ʹ-phosphorylated for subsequent ligation by T4 DNA Ligase (cat. no. L6030-HC).

The high-concentration formulation of the End-Repair Mix is compatible with applications requiring >1 µg of DNA to be prepared for blunt-end ligation.

The low concentration formulation of the End-Repair Mix is compatible with applications requiring <1 µg of DNA to be prepared for blunt-end ligation

Ask us about other concentrations.

This enzyme mix is supplied in 100 mM KCl, 10 mM Tris-HCl, 0.1 mM EDTA, 1 mM DTT, 0.1% Triton X-100 and 50% glycerol: pH 7.4 at 25°C.

10X End-Repair Buffer (cat. no. B9140) contains 1 M Tris-HCl, 500 mM NaCl, 100 mM MgCl₂, 50 mM DTT and 0.25% Triton-X 100; pH 7.5 at 25°C.

SDS available upon request.

Enzyme properties

• Storage temperature: –25°C to –15°C

Source of recombinant enzyme protein
The proteins are produced by strains of E. coli that express the recombinant T4 DNA Polymerase and T4 Polynucleotide Kinase genes, respectively.

1. Prepare purified DNA that is blunted and dissolved in TE buffer (10 mM Tris, 1 mM EDTA; pH 8.0).
2. Combine and mix the following components in a sterile tube:
   • 1–19 µl purified DNA (up to 5 µg)
   • 2.5 µl 10X End-Repair Buffer
   • 2.5 µl 1 mM dNTP mix (cat. no. N2060)
   • 1–3 µl End-Repair Enzyme Mix
   • Sterile water to 25 µl
     Total volume: 25 µl
3. Incubate room temperature (25°C) 30 minutes. Inactivate End-Repair Enzyme by heat at 75°C for 20 minutes.
4. Ligation may be performed immediately using Enzymatics Rapid format T4 DNA Ligase (L6030-HC).

Notes
It is not necessary to add ATP, as the T4 Polynucleotide Kinase contained in the reaction mix uses the deoxynucleotides provided (dATP and dTTP) as phosphate donors.

Quality control analysis

Physical purity is evaluated by SDS-PAGE of concentrated and diluted enzyme solutions followed by silver-stain detection. Purity is assessed by comparing the aggregate mass of contaminant bands in the concentrated sample to the mass of the band corresponding to the protein of interest in the diluted sample.

Functional assays were performed as follows. Two µl End-Repair Mix was added to 1X reaction buffer containing 0.1mM dNTPs and plasmid DNA that had been digested with two restriction enzymes and dephosphorylated. The mixture was incubated at 25°C for 30 minutes. Competent cells were transformed with the reaction mixture, plated onto media containing LB, Amp and X-Gal 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.

Double-stranded endonuclease activity is determined in a 50 µl reaction containing 0.5 µg of plasmid DNA and 10 µl of enzyme solution incubated for 4 hours at 37°C.

E. coli contamination is evaluated using 5 µl replicate samples of enzyme solution that are denatured and screened in a TaqMan qPCR assay for the presence of contaminating E. coli genomic DNA using oligonucleotide primers corresponding to the 16S rRNA locus.

This OEM by QIAGEN product is available for bulk purchase for the following commercial assay applications.

• Create blunt-ended DNA