The End-Repair mix converts DNA containing damaged or incompatible 5′- and/or 3′-protruding ends to 5′-phosphorylated, blunt-ended DNA. This high-concentration formulation of the End-Repair Mix is compatible with applications requiring >1 microgram of DNA to be prepared for blunt-end ligation. 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 (L6030-HC).
Source of Protein
Purified from strains of E. coli that express the recombinant T4 DNA Polymerase, and T4 Polynucleotide Kinase genes, respectively.
100 mM KCl
10 mM Tris-HCl
0.1 mM EDTA
1 mM DTT
0.1% Triton X-100
pH 7.4 @ 25°C
B9140 (10X End-Repair Buffer)
N2060 (1 mM dNTPs)
10X End-Repair Buffer (B9140):
1 M Tris-HCl
500 mM NaCl
100 mM MgCl2
50 mM DTT
0.25% Triton-X 100
pH 7.5 @ 25°C
|Storage Temperature||-25 to -15°C|
|3′→ 5′ Nuclease||Functional|
|5′→ 3′ DNA Synthesis||Functional|
|DS Endonuclease||10 µL = No conversion|
|E.coli DNA Contamination||10 µL <10 copies|
Purified free of contaminating endonucleases. In addition, >99% enzyme purity is analyzed by SDS-PAGE, and negligible E.coli genomic DNA is confirmed by qPCR.
2µL of End-Repair Mix was added to a double restriction enzyme digested, dephosphorylated plasmid DNA in 1X reaction buffer containing 0.1mM dNTPs and incubated at 25°C for 30 minutes. Competent cells were transformed with the ligation mixure, 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
- Purify DNA to be blunted, dissolve in TE buffer.
- 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 (N2060)
1-3 µL End-Repair Enzyme Mix
Sterile H2O to 25 µL
Total Volume: 25 µL
- Incubate room temperature (25°C) 30 minutes. Inactivate End-Repair Enzyme by heat at 75°C for 20 minutes.
- Ligation may be performed immediately using Enzymatics Rapid format T4 DNA Ligase (L6030-HC).
ATP is not required because the T4 Polynucleotide Kinase can utilize the deoxynucleotides (dATP and dTTP) used in the reaction as phosphate donors.
Limitations of Use
This product was developed, manufactured, and sold for in vitro use only. The product is not suitable for administration to humans or animals. SDS sheets relevant to this product are available upon request.