New England Biolabs, M0201S, T4 Polynucleotide Kinase

Related Categories DNA Labeling,, Kinases Applications Phosphorylation (Kinase) Specification Unit Definition One unit of enzyme catalyzes the phosphorylation of 20 pmol of fluorescently labeled oligo in 30 min at 37˚C. 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 DTT (pH 7.6 @ 25°C) Usage Concentration 10,000 units/ml Storage Buffer 10 mM Tris-HCl 50 mM KCl 1 mM DTT 0.1 mM EDTA 50% Glycerol 0.1 µM ATP pH 7.4 @ 25°C Heat Inactivation 65°C for 20 minutes FAQ Q: What factors can cause incomplete phosphorylation when using T4 Polynucleotide Kinase? A: The most likely cause of incomplete phosphorylation is oxidized DTT in the T4 Polynucleotide Kinase (PNK) reaction buffer (DTT oxidation occurs naturally and is acclerated by repeated freeze/thaw cycles or excessive heating). Use fresh buffer (< 1 year) or add fresh DTT to 5 mM using a 1M stock. Other factors include: a. An excess of salt, phosphate or ammonium ions present: PNK is inhibited by high levels of salt (50% inhibition by 150 mM NaCl), phosphate (50% inhibition by 7 mM phosphate) and ammonium ions (75% inhibited by 7 mM (NH4)2SO4). NEB's ThermoPol reaction buffer contains 10 mM (NH4)2SO4 which must be removed before performing a kinase reaction. DNA should not be precipitated in the presence of ammonium ions prior to phosphorylation. Drop dialyze or use a commercially available spin column to remove salt from the sample. b. Small nucleic acid contaminants in the DNA preparation: It is important that the substrate be purified by size selection to remove low molecular weight nucleic acid contaminants which can represent a high percentage of total 5´-OH ends, even if the total mass is low. Use a commercially available spin column to remove these contaminants as well as excess salt. c. The end is recessed or blunt: If the ends are blunt-ended or 5´-recessed, heat the substrate/buffer mixture for 10 minutes at 70°C, chill rapidly on ice before adding the ATP and enzyme, then incubate at 37°C. This helps the PNK access the 5´-OH termini. d. The phosphatase was not inactivated: To remove CIP or BAP use phenol/CHCl3. Quick CIP, Antarctic Phosphatase and rSAP can be heat inactivated. e. ATP not added: PNK requires ATP for activity. Typically, PNK reactions are followed by a ligation reaction. To simplify this process, PNK is optimized for use in T4 Ligase reaction buffer (which contains the appropriate amount of ATP). We recommend performing the PNK reaction in Ligase buffer for 30 minutes; you can then proceed to ligation without a buffer change or heat inactivation; however PNK will remain active in the subsequent reaction unless heat inactivated or removed (spin column, gel purification) prior to the next step. Q: Can I use T4 Polynucleotide Kinase and T4 DNA Ligase in the same reaction buffer? A: Yes, for non-radioactive phosphorylations, but not for radioactive labeling reactions since the non-radioactive ATP in T4 Ligase buffer will interfere with labeling. Q: How can the rate of phosphorylation be improved when using T4 Polynucleotide Kinase? A: a. If working with 5´-recessed ends, heat the reaction mixture for 10 min at 70°C, chill rapidly on ice before adding the ATP (or Ligase buffer containing ATP) and enzyme, then incubate at 37°C. b. Add PEG to the reaction. The addition of PEG 8000 to 5% final (w/v) can improve the results. c. Add spermidine. Spermidine will enhance the reactions approximately 20-30%, but it is not used in the unit determination and is not required for full activity. d. Use an alternative buffer for the exchange reaction. Higher levels of incorporation in the exchange reaction can be attained by using a buffer containing 50 mM imidazole-Cl (pH 6.4), 10 mM MgCl2, and 5 mM DTT (Sambrook, J. et al. (1989) Molecular Cloning, second edition, pp 10.59-10.67, 11.31-11.33, Cold Spring Harbor Laboratory, Cold Spring Harbor). This buffer is not supplied by NEB. Q: Do I need to dephosphorylate prior to labeling? A: Not necessarily. The exchange reaction gives acceptable results, but dephosphorylating first, followed by the forward reaction, gives the best incorporation. Q: How much substrate can be phosphorylated in a standard reaction? A: Up to 350 pmol of 5´ termini for cold phosphorylation and up to 50 pmol for hot phosphorylation. Note: 1 µg of a 20 mer = 150 pmol of 5´ termini. Example, to calculate the number of pmol in 1 µg of a 30 mer: 150 pmol X (20 mer/30 mer) = 100 pmol Q: How many units of T4 Polynucleotide Kinase should be used for a typical reaction? A: Typically, 10 units for a cold reaction and 20 units for a hot labeling reaction. Since the [substrate] and the [ATP] are below the Km in a hot reaction, more units are required. The unit determination is done under ideal conditions where the substrate and ATP concentrations are above their Km's. Q: How do I inactivate the enzyme? A: Incubation for 20 minutes at 65°C completely inactivates T4 Polynucleotide Kinase. Q: How to calculate the molarity of ends? A: NEB suggests using the free online tool, NEBioCalculator. This tool will calculate molarity of ends as well as other biomath calculations. If you would like to calculate “by hand”, please note the following: Molarity = [(µg/µl) ÷ (base pairs x 650 daltons)] x 2 ends Example: Calculate the molarity of ends for a linearized 5 kb vector that has a concentration of 250 ng/µl: [(0.25 µg/µl) ÷ (5000 x 650 daltons)] x 2 = 154 nM Calculate the molarity of ends if you put 50 ng of this vector in a 20 µl ligation reaction: 50 ng ÷ 20 µl = 0.0025 µg/µl [(0.0025 µg/µl) ÷ (5000 x 650)] x 2 = 1.54 nM Determine the amount of a 1 kb insert needed to achieve a 3:1 insert:vector ratio: 3 X 1.54 nM = 4.62 nM [(X µg/µl) ÷ (1000 x 650)] x 2 = 4.62 nM 0.0015 µg/µl x 20 µl = 0.03 µg = 30 ng Does this reaction fall within the optimal range of 1–10 µg/ml? (50 ng vector + 30 ng insert) ÷ 20 µl = 4 µg/ml Q: What labels can be used? A: Phosphorylate with either 32P-ATP or 33P-ATP (35S does not work as a donor). Due to the low energy emissions of 33P-ATP, films must be exposed for extended periods. Scintillation measurements work well with 33P-ATP. Q: Will T4 Polynucleotide Kinase work in rCutSmart Buffer? A: T4 Polynucleotide Kinase will work in rCutSmart Buffer, with the addition of ATP and DTT. To see its % functional activity in rCutSmart, and that of other DNA Modifying enzymes in the cloning workflow, refer to the Activity of DNA Modifying Enzymes in rCutSmart® Buffer chart.