T4 DNA Polymerase
T4 DNA Polymerase catalyzes the 5'→3' incorporation of nucleotides into double-stranded DNA using single-stranded and primed DNA as a template. It possesses strong 3'→5' exonuclease (proofreading) activity but does not exhibit 5'→3' exonuclease activity. Typical T4 DNA polymerase protocols include 5' DNA overhang blunting as well as the generation of blunt double-stranded DNA from double-stranded DNA containing 3' overhangs. These DNA products are often used in blunt cloning. T4 DNA Polymerase is supplied in a buffer of 200 mM potassium phosphate (pH 6.5), 10 mM DTT and 50% glycerol.
- Generation of blunt double-stranded DNA from DNA containing 5'/3' overhangs
- 5'-end or 3'-end labeling of double-stranded DNA
- In vitro mutagenesis
Recombinant E. coli
Supplied with 10X Reaction Buffer [330 mM Tris-acetate (pH 7.9), 660 mM potassium acetate, 100 mM magnesium acetate and 5 mM DTT] and 0.1% BSA.
One unit is defined as the amount of enzyme that catalyzes the incorporation of 10 nmol of total nucleotides into acid-insoluble product in 30 minutes at 37°C and pH 8.8 using heat-denatured calf thymus DNA as the template-primer.
Butler, E. T. & Chamberlins, M. J. Bacteriophage SP6-specific RNA Polymerase I. ISOLATION AND CHARACTERIZATION OF THE ENZYME*. J. Biol. Chem. 257, 5772–5778 (1982).
Green, M. R., Maniatis, T. & Melton, D. A. Human β-globin pre-mRNA synthesized in vitro is accurately spliced in Xenopus oocyte nuclei. Cell 32, 681–694 (1983).
Kotaoi, H., Ishizalci, Y., Hiraoka, N. & Obayashi, A. Nucleotide sequence and expression of the doned gene of bacteriophage SP6 RNA polymerase. Nucleic Acids Res. 15, (1987).
Melton, D. A. et al. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 12, 7035–56 (1984).
Pelletier, J. & Sonenberg, N. Insertion mutagenesis to increase secondary structure within the 5′ noncoding region of a eukaryotic mRNA reduces translational efficiency. Cell 40, 515–526 (1985).
Sagawa, H., Ohshima, A. & Kato, I. A tightly regulated expression system in Escherichia coli with SP6 RNA polymerase. Gene 168, (1996).
Schenborn, E. T. & Mierendorf, R. C. A novel transcription property of SP6 and T7 RNA polymerases: dependence on template structure. Nucleic Acids Res. 13, 6223–36 (1985).
Additional product information
Please see the product's Certificate of Analysis for information about storage conditions, product components, and technical specifications. Please see the Kit Components List to determine kit components. Certificates of Analysis and Kit Components Lists are located under the Documents tab.
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