End-Repair Mix (Low concentration)

Catalog Number KEM0010

Storage Temperature –20 °C
Unit Size 200 Reactions

Product Description

The End-Repair Mix converts DNA containing damaged or incompatible 5′- and/or 3′-protruding ends to 5′-phosphorylated, blunt-ended DNA. This low-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, Catalog Number KEM0033. T4 Polynucleotide Kinase, Catalog Number KEM0006, ensures that the ends of the blunt-ended DNA fragments are 5′-phosphorylated for subsequent ligation by T4 DNA Ligase, Catalog Number KEM0019.

Source of Protein

Purified from strains of E. coli that express the recombinant T4 DNA Polymerase, and T4 Polynucleotide Kinase genes, respectively.

Reagent

Supplied at a concentration of 1 Rxn/µL 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

Supplied with:

1 mM dNTP Mix
Catalog Number KEM0069B

10X End-Repair Buffer
Catalog Number KEM0065B
1 M Tris-HCl, 500 mM NaCl, 100 mM MgCl2,
50 mM DTT, 0.25% Triton™ X-100, pH 7.5

Precautions and Disclaimer

This product is for R&D use only, not for drug, household, or other uses. Please consult the Safety Data Sheet for information regarding hazards and safe handling practices.

Protocol

1.     Purify DNA to be blunted, dissolve in TE buffer.
2.     Combine and mix the following components in a sterile tube:

Component Volume (µL)
Purified DNA (up to 1µg) 1-19 µL
10X End-Repair Buffer 1x 2.5 µL
1 mM dNTP Mix 2.5 µL
End-Repair Mix 1-3 µL
Sterile Water X µL
Total Volume 25 µL

3.     Incubate at room temp (25 °C) for 30 minutes.  Inactivate End-Repair enzyme by heat at 75 °C for 20 minutes.
4.     Ligation may be performed immediately using T4 DNA Ligase, Catalog Number KEM0019.

Usage Notes

ATP is not required because the T4 Polynucleotide kinase can utilize the deoxynucleotides (dATP and dTTP) used in the reaction as phosphate donors.

Triton is a trademark of The Dow Chemical Company or an affiliated company of Dow.

 

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