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T4 Polynucleotide Kinase > FAQ |  | T4 Polynucleotide Kinase FAQ
See the DNA Modifying Enzymes and Cloning FAQ also.
Q1: What factors can cause incomplete phosphorylation when using T4 Polynucleotide Kinase?
Q2: Can I use T4 Polynucleotide Kinase and T4 DNA Ligase in the same reaction buffer?
Q3: How can the rate of phosphorylation be improved when using T4 Polynucleotide Kinase?
Q4: Do I need to dephosphorylate prior to labeling?
Q5: How much substrate can be phosphorylated in a standard reaction?
Q6: How many units of T4 Polynucleotide Kinase should be used for a typical reaction?
Q7: How do I inactivate the enzyme?
Q8: What labels can be used?
Q1: What factors can cause incomplete phosphorylation when using T4 Polynucleotide Kinase?
A1: 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. Antarctic Phosphatase and SAP 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.
Q2: Can I use T4 Polynucleotide Kinase and T4 DNA Ligase in the same reaction buffer?
A2: Yes, for cold phosphorylations, but not for hot labeling reactions since the cold ATP in T4 Ligase buffer will interfere with labeling.
Q3: How can the rate of phosphorylation be improved when using T4 Polynucleotide Kinase?
A3: 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.
Q4: Do I need to dephosphorylate prior to labeling?
A4: Not necessarily. The exchange reaction gives acceptable results, but dephosphorylating first, followed by the forward reaction, gives the best incorporation.
Q5: How much substrate can be phosphorylated in a standard reaction?
A5: 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
Q6: How many units of T4 Polynucleotide Kinase should be used for a typical reaction?
A6: 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.
Q7: How do I inactivate the enzyme?
A7: Incubation for 20 minutes at 65°C completely inactivates T4 Polynucleotide Kinase.
Q8: What labels can be used?
A8: 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.
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