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T4 Polynucleotide Kinase

#EK0031 Supplied with: 10X Reaction Buffer A 10X Reaction Buffer B 24% PEG Solution	500 u (10 u/µl) 0.4 ml 0.2 ml 0.2 ml
#EK0032 Supplied with: 10X Reaction Buffer A 10X Reaction Buffer B 24% PEG Solution	2500 u (10 u/µl) 2 ml 1 ml 1 ml
Related Documents (in pdf, ~40-60 KB): Certificate of Analysis: #EK0031, #EK0032 MSDS (English) MSDS (English-USA) MSDS (German)

 

Feature
Active in Fermentas buffers for restriction enzymes, PCR and RT.

Description
The T4 Polynucleotide Kinase (T4 PNK) is a polynucleotide 5'-hydroxyl kinase that catalyzes the transfer of the g-phosphate from ATP to the 5'-OH group of single- and double-stranded DNAs and RNAs, oligonucleotides or nucleoside 3'-monophosphates (forward reaction). The reaction is reversible. In the presence of ADP T4 Polynucleotide Kinase exhibits 5'-phosphatase activity and catalyzes the exchange of terminal 5'-phosphate groups (exchange reaction) (1).
The enzyme is also a 3'-phosphatase (2).

Source
E.coli cells with a cloned pseT gene of bacteriophage T4.

Molecular Weight
The enzyme is a homotetramer. It consists of four identical subunits of 28.9 kDa.

Labeling efficiency of the DNA containing different types of 5'-ends.

Applications

• Labeling 5'-termini of nucleic acids by either the forward or the exchange reaction (3, 4), see picture on the right and Protocol for Labeling 5'-termini of DNA by Forward Reaction, Protocol for Labeling 5'-protruding Termini of DNA by Exchange Reaction and Protocol for Phosphorylation of 5'-termini of DNA or Oligonucleotide.
  The 5'-labeled nucleic acids can be used as:
  • probes for hybridization,
  • probes for transcript mapping,
  • markers for gel-electrophoresis,
  • primers for DNA sequencing,
  • primers for PCR.
• 5'-phosphorylation of oligonucleotide linkers and DNA or RNA prior to ligation.
• Detection of DNA modification by the [³²P]-postlabeling assay (5, 6).
• Removal of 3'-phosphate groups (2).

Quality Control
Tested for the absence of endo-, exodeoxyribonucleases, ribonucleases and for labeling 5'-termini of DNA.

Concentration
10 u/µl

Definition of Activity Unit
One unit of the enzyme transfers 1 nmol of gama-phosphate from ATP to 5'-OH DNA in 30 min at 37°C.
Enzyme activity is assayed in the following mixture: 100 mM Tris-HCl (pH 8.0), 10 mM MgCl₂, 5 mM DTT, 0.5 mM 5'-OH DNA, 0.05 mM ATP and 0.1 MBq/ml [gama-³³P]-ATP.

Storage Buffer
The enzyme is supplied in: 20 mM Tris-HCl (pH 7.5), 25 mM KCl, 0.1 mM EDTA, 2 mM DTT and 50% (v/v) glycerol.

10X Reaction Buffer A (for forward reaction)
500 mM Tris-HCl (pH 7.6 at 25°C), 100 mM MgCl₂, 50 mM DTT, 1 mM spermidine and 1 mM EDTA.

10X Reaction Buffer B (for exchange reaction)
0.5 M imidazole-HCl (pH 6.4 at 25°C), 0.18M MgCl₂, 50 mM DTT, 1 mM spermidine, 1 mM EDTA and 1 mM ADP.

24% PEG Solution
24% (w/v) polyethylene glycol 6000.

Inhibition and Inactivation

• Inhibitors: metal chelators, phosphate and ammonium ions, KCl and NaCl at a concentration higher than 50 mM.
• Inactivated by heating at 75°C for 10 min or by the addition of EDTA.

Note

• Polyethylene glycol (PEG) and spermidine improve the rate and efficiency of the phosphorylation reactions (7). PEG should be added to the exchange reaction mixture, see Protocol for Labeling 5'-protruding Termini of DNA by Exchange Reaction.
• As T4 Polynucleotide Kinase is inhibited by ammonium ions, DNA precipitation is recommended in the presence of sodium acetate (3, 4).

Related Products

T4 Polynucleotide Kinase Calf Intestine Alkaline Phosphatase (CIAP) Shrimp Alkaline Phosphatase (SAP) ATP 0.5 M EDTA, pH 8.0 Water, nuclease-free DEPC-treated Water

References

1. Berkner, K.L., Folk, W.R., Polynucleotide kinase exchange reaction, J. Biol. Chem., 252, 3176-3184, 1977.
2. Richardson, C.C., Bacteriophage T4 polynucleotide kinase, The Enzymes (Boyer, P.D., ed.), 14, 299-314, Academic Press, San Diego, 1981.
3. Sambrook, J., Russell, D.W., Molecular Cloning: A Laboratory Manual, the third edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001.
4. Current Protocols in Molecular Biology, vol. 1 (Ausubel, F.M., et al., ed.), John Wiley & Sons, Inc., Brooklyn, New York, 3.10.2-3.10.5, 1994-2005.
5. Phillips, D.H., Detection of DNA modifications by the ³²P-postlabelling assay, Mutation Res., 378, 1-12, 1997.
6. Keith, G., Dirheimer, G., Postlabeling: a sensitive method for studying DNA adducts and their role in carcinogenesis, Curr. Opin. Biotechnol., 6, 3-11, 1995.
7. Harrison, B., Zimmerman, S.B., T4 polynucleotide kinase: macromolecular crowding increases the efficiency of reaction at DNA termini, Anal. Biochem., 158, 307-315, 1986.

Updated kovo 18, 2008 10:05