T4 Polynucleotide Kinase Catalog # Size Concentration M0201S   STOCK! 500 units 10,000 units/ml M0201L 2,500 units 10,000 units/ml Description: Catalyzes the transfer and exchange of Pi from the γ position of ATP to the 5´ -hydroxyl terminus of polynucleotides (double-and single-stranded DNA and RNA) and nucleoside 3´-monophosphates. Polynucleotide Kinase also catalyzes the removal of 3´-phosphoryl groups from 3´-phosphoryl polynucleotides, deoxynucleoside 3´-monophosphates and deoxynucleoside 3´-diphosphates (1). Source: A E. coli strain that carries the cloned T4 Polynucleotide Kinase gene. It is purified by a modification of the method of Richardson (1). Applications: End-labeling DNA or RNA for probes and DNA sequencing (2) Addition of 5´-phosphates to oligonucleotides to allow subsequent ligation Removal of 3´-phosphoryl groups (3) Reagents Supplied: T4 Polynucleotide Kinase Reaction Buffer (10X) Enzyme Properties 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 Richardson unit is defined as the amount of enzyme catalyzing the incorporation of 1 nmol of acid-insoluble [32P] in a total reaction volume of 50 μl in 30 minutes at 37°C in 1X T4 Polynucleotide Kinase Reaction Buffer with 66 µM [γ-32P] ATP (5 x 106 cpm/µmol) and 0.26 mM 5´-hydroxyl-terminated salmon sperm DNA (1). Concentration: 10,000 units/ml Storage Conditions: 10 mM Tris-HCl 50 mM KCl 0.1 µM ATP 1 mM Dithiothreitol 0.1 mM EDTA 50% Glycerol pH 7.4 @ 25°C Storage Temperature: -20°C Notes 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 Buffer, 50 pmol of gamma-[32]P ATP and 20 units of T4 Polynucleotide Kinase. For non-radioactive phosphorylation use up to 300 pmol of 5´ termini in a 50 µl reaction containing 1X T4 Polynucleotide Kinase Buffer, 1 mM ATP and 10 units of T4 Polynucleotide Kinase. Incubate at 37°C for 30 minutes. 1X T4 DNA Ligase 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 (the reduced dithiothreitol in older buffer 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 labelling (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 but not nicks can be labeled with elevated levels of ATP (1). T4 Polynucleotide Kinase Reaction Buffer is suitable for forward reactions, but for the purpose of radiolabeling does not contain ATP. For nonradioactive phosphorylation, supplement to a final ATP concentration of 1 mM or use T4 DNA Ligase Buffer.