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DESCRIPTION
T4 Polynucloetide Kinase catalyzes the transfer of the γ-phosphate of ATP to a 5'-terminus of polynucleotides (double-stranded and single-stranded DNA or RNA) and nucleoside 3'-monophosphates. In the forward reaction, the γ-phosphate is transferred to the 5'-terminus of dephosphorylated DNA. In the exchange reaction, an excess of ADP causes T4 polynucleotide kinase to transfer the terminal 5'-phosphate from phosphorylated DNA to ADP, the DNA is then rephosphorylated by transfer of a radiolabeled γ-phosphate from [γ-32P]ATP. This enzyme also contains a 3'-phosphatase activity.
SOURCE
Recombinant strain of E. coli.
APPLICATIONS
● 5' end labeling of single- or double-stranded DNA and RNA for probes and for DNA sequencing.
● Addition of 5'-phosphates to synthetic linkers or DNA fragments that lack terminal 5'-phosphates for subsequent ligation
UNIT DEFINITION
One unit is defined as the amount of enzyme required to catalyze the transfer of 1 nmol phosphate from [γ-32P]ATP to the 5'-OH terminus of an oligonucleotide in 30 minutes at 37℃.
STANDARD UNIT ASSAY CONDITIONS
70mM Tris-HCl (pH7.6), 10mM MgCl2, 5mM DTT, 66 μM [γ-32P]ATP, 0.26mM 5'-hydroxyl terminated salmon sperm DNA.
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CONCENTRATION: 5-10 units/μl.
10X REACTION BUFFER
700mM Tris-HCl (pH7.6@37℃), 100mM MgCl2, 50mM DTT. Incubate at 37℃.
STORAGE CONDITIONS
10mM Tris-HCl (pH 7.4), 50mM KCl, 0.1mM EDTA, 1mM DTT, 0.1 μM ATP and 50% glycerol. Store at -20℃.
NOTES
1. ATP should be present at a concentration of at least 1μM in the forward reaction and at least 2μM in the exchange reaction, but optimal activity occurs at higher concentrations.
2. T4 polynucleotide kinase is strongly inhibited by ammonium ions. Therefore DNA should not be dissolved in, or precipitated from, buffers containing ammonium ions prior to phosphorylation.
REFERENCES
1. Richardson, C. C. (1981) In The Enzymes (Boyer, P. D., ed.) Vol. 14, pp 299-314, Academic Press.
2. Maxam, A. M. and Gilbert, W. (1980) Methods Enzymol. 65, 499.
3. Richardson, C. C. (1972) Nucleic Acids Res. 2, 815.
4. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, second ed., Cold Spring Harbor Laboratory, Cold Spring Harbor.
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