M.B. Enzymes GmbH

T4 polynucleotide kinase.

Description

T4 PNK (EC 2.7.1.78) catalyzes the transfer of a g-phosphate group from a 5´-nucleoside triphosphate moiety to a free 5´-hydroxyl of a polynucleotide such as DNA or RNA molecule and a nucleoside-3-monophosphate or deoxynucleoside-3-monophosphate. Excess of a nucleotide diphosphate such as ADP will cause the reverse reaction to be favored. That is used in phosphate exchange reaction for labeling of the DNA or RNA ends.

Unit definition

One unit of the enzyme catalyzes the incorporation of 1 nmol of phosphate to the 5´-OH end of an oligonucleotide from (g-³²P)ATP in 30 min at 37°C. The enzyme has specific activities in the range of 30.000-40.000 U/mg.

Reaction buffers

Tris Kinase Buffer B11 (x10): 500 mM TrisHCl 7.6, 100 mM MgCl₂, 50 mM DTT, 1 mM spermindine, 1 mM EDTA.

Imidazole buffer (for kinasing or phosphate exchange) B12 (x10): 500 mM Imidazole-HCl 6.4, 180 mM MgCl₂, 50 mM DTT, 1 mM spermidine, 1 mM EDTA.

Reaction conditions

The phosphate group donor for the kinase reaction may be any nucleoside triphosphates at concentration >1 µM. A total of 1.7 mM of spermine can increase reaction rate by 30-fold. Per reaction use 10-20 U PNK, 1-50 pmol DNA or 10 pmol of oligonucleotide, and 20 pM (g-³²P)ATP (end concentration of ATP should be at least 1µM).

Tris kinase buffer is used for kinasing of ssDNA fragments, duplexes with protruding 5´ termini or labeling of oligonucleotides; the imidasole buffer is used for exchnage reaction and kinasing DNA duplexes with blunt or recessed 5´ ends. For exchange reaction, ADP and ATPshould be added at a final conc. of 0,1 mM and 1 nM, respectively.

Storage

Store at -20°C.

Applications

phosphorylation of the 5´ termini and end labeling of DNA, RNA or synthetic oligonucleotides, removal of 3´-phosphoryl groups.

Quality control

activity, SDS-PAGE purity, absence of endo- and exonucleases, specific performance tests.

References

1. Richardson, C.C. (1965) Proc. Natl. Acad. Sci. USA 54, 158-163. 2. van de Sande, J.H. et al. (1973) Biochemistry 12, 5050-5055. 3. Lillehaug, J.R. et al. (1976) Biochemistry 15, 1858-1865.

Catalog #

M4.4

M4.5

Pack size

1.000 U

2.500 U

T4 polynucleotide kinase.
Description	T4 PNK (EC 2.7.1.78) catalyzes the transfer of a g-phosphate group from a 5´-nucleoside triphosphate moiety to a free 5´-hydroxyl of a polynucleotide such as DNA or RNA molecule and a nucleoside-3-monophosphate or deoxynucleoside-3-monophosphate. Excess of a nucleotide diphosphate such as ADP will cause the reverse reaction to be favored. That is used in phosphate exchange reaction for labeling of the DNA or RNA ends.
Unit definition	One unit of the enzyme catalyzes the incorporation of 1 nmol of phosphate to the 5´-OH end of an oligonucleotide from (g-³²P)ATP in 30 min at 37°C. The enzyme has specific activities in the range of 30.000-40.000 U/mg.
Reaction buffers	Tris Kinase Buffer B11 (x10): 500 mM TrisHCl 7.6, 100 mM MgCl₂, 50 mM DTT, 1 mM spermindine, 1 mM EDTA. Imidazole buffer (for kinasing or phosphate exchange) B12 (x10): 500 mM Imidazole-HCl 6.4, 180 mM MgCl₂, 50 mM DTT, 1 mM spermidine, 1 mM EDTA.
Reaction conditions	The phosphate group donor for the kinase reaction may be any nucleoside triphosphates at concentration >1 µM. A total of 1.7 mM of spermine can increase reaction rate by 30-fold. Per reaction use 10-20 U PNK, 1-50 pmol DNA or 10 pmol of oligonucleotide, and 20 pM (g-³²P)ATP (end concentration of ATP should be at least 1µM). Tris kinase buffer is used for kinasing of ssDNA fragments, duplexes with protruding 5´ termini or labeling of oligonucleotides; the imidasole buffer is used for exchnage reaction and kinasing DNA duplexes with blunt or recessed 5´ ends. For exchange reaction, ADP and ATPshould be added at a final conc. of 0,1 mM and 1 nM, respectively.
Storage	Store at -20°C.
Applications	phosphorylation of the 5´ termini and end labeling of DNA, RNA or synthetic oligonucleotides, removal of 3´-phosphoryl groups.
Quality control	activity, SDS-PAGE purity, absence of endo- and exonucleases, specific performance tests.
References	1. Richardson, C.C. (1965) Proc. Natl. Acad. Sci. USA 54, 158-163. 2. van de Sande, J.H. et al. (1973) Biochemistry 12, 5050-5055. 3. Lillehaug, J.R. et al. (1976) Biochemistry 15, 1858-1865.
Catalog #	M4.4	M4.5
Pack size	1.000 U	2.500 U