Abstract for DNA 5′ End-Labeling System
T4 Polynucleotide Kinase (PNK) catalyzes the transfer of the terminal [γ-32P]
phosphate of ATP to the 5′-hydroxyl terminus of a DNA molecule. The DNA 5′ End-Labeling System is a complete system for phosphorylating both
oligonucleotides and DNA fragments using T4 PNK. 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 relative weak activity of 35S, much longer exposure times are generally required.
Both an Oligonucleotide and the pGEM® DNA Markers are included as
controls for the efficiency of the reaction. The pGEM® DNA Markers
also provide a convenient range of fragments for analysis of phosphorylated DNA
samples by gel electrophoresis.
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