T4 Polynucleotide Kinase

Source

Recombinant E. coli strain

Reagents Supplied

10X T4 Polynucleotide Kinase Reaction Buffer

Applications

• Catalyzes the transfer of the gamma-phosphate of ATP to the 5'-OH terminus in DNA or RNA
• Contains 3'-phosphatase activity (1)
• Used for 5'-end labeling of nucleic acids prior to DNA or RNA sequencing (2, 3)
• Phosphorylates synthetic linkers and fragments of DNA or RNA prior to ligation
• Labels 5'-termini prior to partial restriction enzyme digestion

Unit Definition

One unit is the amount of enzyme required to transfer 1 nmol of γ-phosphate from ATP to the 5' OH terminus of salmon sperm DNA at 37°C in 30 minutes

Heat Inactivation

15 minutes at 65ºC

Concentration

20 - 30 Units/µl

Storage Buffer

50 mM Tris HCl (pH 7.6)
0.1 mM EDTA
25 mM KCl
5 mM Dithiothreitol
0.1 µM ATP
50% (v/v) Glycerol

Assay Conditions

70 mM Tris-HCl (pH 7.6)
10 mM MgCl₂
5 mM Dithiothreitol
50 ug 5'-OH terminated DNA (micrococcal nuclease treated salmon sperm DNA)
0.07 mM [γ-32P] ATP
Reaction volume of 100 µl

Storage Conditions

Store at -20°C
Product shipped on dry ice