FAQ: How can the rate of phosphorylation be improved when using T4 Polynucleotide Kinase?

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.