DNA 5´ End-Labeling System Technical Bulletin

Literature # TB096

The DNA 5′ End-Labeling System is a complete system for phosphorylating both oligonucleotides and DNA fragments using T4 polynucleotide kinase (PNK), which catalyzes the transfer of the terminal [γ-³²P]phosphate of ATP to the 5′-hydroxyl terminus of a DNA molecule. Calf Intestinal Alkaline Phosphatase (CIAP) is provided. CIAP may be used to remove the 5′ phosphate from DNA fragments, which can be subsequently end-labeled with a radioactive phosphate.

This protocol can be used with either [γ-³²P]ATP or [γ-³³P]ATP for 5′ end-labeling. [γ-³⁵S]ATP can be used for 5′ end-labeling; however, due to the relatively weak activity of ³⁵S, much longer exposure times are generally required. Both an oligonucleotide and the pGEM® DNA Markers are included as controls for labeling efficiency. The pGEM® DNA Markers also provide a convenient range of fragments for analysis of phosphorylated DNA samples by gel electrophoresis.