To access your account, log in or register.	shopping cart | log in

FAQs for DNA Modifying Enzymes and Cloning Technical Reference for DNA Modifying Enzymes and Cloning Enzyme Finder NEBcutter NEBuffer Chart Double Digest Finder Isoschizomers DNA Sequences and Maps REBASE T4 Polynucleotide Kinase Reaction Buffer

Home > Products > DNA Modifying Enzymes and Cloning > Kinase > T4 Polynucleotide Kinase (3' phosphatase minus) T4 Polynucleotide Kinase (3' phosphatase minus) Catalog # Size Concentration Price Qty   M0236L 1,000 units 10,000 units/ml $400.00 M0236S 200 units 10,000 units/ml $100.00 Prices are in US dollars and valid only for US orders. Download: Technical Bulletin|MSDS PDF Isolated from a recombinant source 5' end labeling of DNA and RNA RNase and DNase free Supplied with 10X Reaction Buffer Description: T4 Polynucleotide Kinase catalyzes the transfer and exchange of Pi from the γ position of ATP to the 5´ -hydroxyl terminus of double- and single-stranded DNA and RNA, as well as nucleoside 3´-monophosphates (1-5). This modified version exhibits full kinase activity with no 3´ phosphatase activity (6,7). Source: An E. coli strain that carries a plasmid encoding the modified T4 Polynucleotide Kinase gene. Applications: 5´ phosphorylation of DNA/RNA for subsequent ligation End-labeling of DNA or RNA (2) 5´ phosphorylation of 3´ phosphorylated mononucleotides to generate a substrate (pNp) that can be added to the 3´ end of DNA or RNA by ligase activity (8) 5´ end labeling of 3´ phosphorylated oligos (9) Reagents Supplied: T4 Polynucleotide Kinase Reaction Buffer (10X) Enzyme Properties Protein Kinase Substrate Recognition Heat Inactivation: 65°C for 20 minutes Reaction & Storage Conditions Reaction Conditions: 1X T4 Polynucleotide Kinase Reaction Buffer Incubate at 37°C. 1X T4 Polynucleotide Kinase Reaction Buffer: 70 mM Tris-HCl 10 mM MgCl2 5 mM Dithiothreitol pH 7.6 @ 25°C Unit Definition: One unit is defined as the amount of enzyme catalyzing the incorporation of 1 nmol of acid-insoluble [32P] in 30 minutes at 37°C (1).  Unit Assay Conditions:  1X T4 Polynucleotide Kinase Reaction Buffer, 66 µM [γ-32P] ATP (5 x 106 cpm/µmol), 0.26 mM 5´-hydroxyl-terminated salmon sperm DNA. Concentration: 10,000 units/ml Storage Conditions: 10 mM Tris-HCl 50 mM KCl 1 mM DTT 0.1 µM ATP 0.1 mM EDTA 50% Glycerol pH 7.4 @ 25°C Storage Temperature: -20°C Notes General notes: T4 Polynucleotide Kinase requires ATP for activity, but its supplied reaction buffer does not contain ATP because it interferes with radiolabeling reactions. Typically, a kinase reaction is followed by a ligation reaction. To simplify this process, T4 Polynucleotide Kinase is optimized for use in T4 DNA Ligase reaction buffer (which contains the appropriate amount of ATP). We recommend performing the kinase reaction in ligase buffer for 30 minutes; you can then proceed to ligation without a buffer change or heat inactivation. Usage notes: [33P] ATP may be substituted for [32P] ATP. For radioactive labeling, use 1-50 pmol of 5´ termini in a 50 µl reaction containing 1X T4 Polynucleotide Kinase Reaction Buffer, 50 pmol of gamma-[32P] ATP and 20 units of T4 Polynucleotide Kinase. Incubate at 37°C for 30 minutes. For non-radioactive phosphorylation use up to 300 pmol of 5´ termini in a 50 µl reaction containing 1X T4 Polynucleotide Kinase Reaction Buffer, 1 mM ATP and 10 units of T4 Polynucleotide Kinase. Incubate at 37°C for 30 minutes. 1X T4 DNA Ligase Reaction Buffer contains 1 mM ATP and can be substituted in non-radioactive phosphorylations (T4 Polynucleotide Kinase exhibits 100% activity in this buffer). Fresh buffer is required for optimal activity (in older buffers, loss of DTT due to oxidation lowers activity). The efficiencies of blunt and recessed 5´-end phosphorylation can be improved by heating to 70°C for 5 minutes, then chilling on ice prior to kinase addition and by adding PEG-8,000 to 5% (w/v) (2). The following levels of inhibition of T4 Polynucleotide Kinase are observed when the supplied reaction buffer is supplemented with: 50 mM NaCl - no inhibition 100 mM NaCl - 30% inhibition 150 mM NaCl - 50% inhibition 7 mM phosphate - 50% inhibition 7 mM (NH4)2SO4 - 75% inhibition  Since Polynucleotide Kinase is inhibited by ammonium ions, DNA should not be precipitated in the presence of ammonium ions prior to phosphorylation. Dephosphorylation prior to end-labeling can be avoided by utilizing the exchange reaction, although this results in lower specific activity labeling (4). Sufficient incorporation levels can be attained using the supplied buffer supplemented with 100 µM ADP in the final reaction. However, higher levels of incorporation with the exchange reaction are achieved when using the buffer described in (2). Gaps can be phosphorylated with elevated levels of ATP. Nicks are not phosphorylated efficiently. CTP, GTP, TTP, UTP, dATP or dTTP can be substituted for ATP as a phosphate donor (1). Quality Control for Current Lot Quality control values for a specific lot can be found on the datacard which accompanies each product. Quality Assurance Statement: Free of exonuclease, phosphatase, endonuclease and RNase activities. Each lot is tested under 5´-end-labeling conditions to assure maximal transfer of [32P]. 16-Hour Incubation: A 50 μl reaction containing 1 μg of λDNA and 150 units of T4 Polynucleotide Kinase (3' phosphatase minus) incubated for 16 hours at 37ºC resulted in a DNA pattern free of detectable nuclease degradation as determined by agarose gel electrophoresis. Exonuclease Activity: Incubation of a 50 μl reaction containing 100 units of T4 Polynucleotide Kinase (3' phosphatase minus) with 1 μg of a mixture of single and double-stranded [3H] E. coli DNA (205 cpm/μg) for 4 hours at 37ºC released < 0.1% of the total radioactivity. Endonuclease Activity: Incubation of a 50 μl reaction containing 100 units of T4 Polynucleotide Kinase (3' phosphatase minus) with 1 μg of ΦX174 RF I DNA for 4 hours at 37ºC resulted in < 10% conversion to RFII as determined by agarose gel electrophoresis. RNase Assay: Incubation of a 50 μl reaction containing 50 units of T4 Polynucleotide Kinase (3' phosphatase minus) with 2 μg of MS2 RNA for 1 hour at 37ºC resulted in no detectable degradation of the RNA as determined by agarose gel electrophoresis. Other Activities: 32P end labeling of 5´-hydroxyl terminated d(T)8 with 25 units for 30 minutes at 37°C in 50 µl 1X T4 Polynucleotide Kinase Reaction Buffer followed by 20% acrylamide gel electrophoresis revealed that less than 1% of the product had been degraded by exonuclease or phosphatase activities. References Richardson, C.C. (1981) In P.D. Boyer (Ed.). The Enzymes, 14, 299-314. San Diego: Academic Press. Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, (2nd ed.), 10.59-10.67, 11.31-11.33. Cold Spring Harbor: Cold. Cameron, V. and Uhlenbeck, 0.C. (1977) Biochemistry, 16, 5120-5126. Berkner, K.L. and Folk, W.R. (1977) J. Biol. Chem., 252, 3176-3184. Soltis, D.A. and Uhlenbeck, O.C. (1982) J. Biol. Chem., 257, 11332-11339. Wang, L.K. and Shuman, S. (2001) J. Biol. Chem., 276, 26868-26874. Wang, L.K. and Shuman, S. (2002) Nucl. Acids Res., 30, 1073-1080. Vance, J.R. and Wilson, T.E. (2001) Mol. Cell. Biol., 21, 7191-7198. Interthal, H. et al. (2005) J. Biol. Chem., 280, 36518-36528. Reagents Sold Separately T4 Polynucleotide Kinase Reaction Buffer