For 200 low-concentration DNA reactions. End-Repair Mix 1X (0.20 mL), 10X End-Repair Buffer (1 x 1.5 mL) and 1 mM dNTP solution (0.5 mL).
The End-Repair Mix is intended for molecular biology applications. This product is neither intended for the diagnosis, prevention or treatment of a disease, nor has it been validated for such use either alone or in combination with other products.
Features
- 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
Product Details
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.
Performance
| 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 |