Optimization of conditions for labeling the 3' OH end of tRNA using T4 RNA ligase

For several years most primary structure studies of ribonucleic acids have used the 32P] in vitro post-labeling techniques. We adapted our methods from the literature, and simplified them to make them accessible to any laboratory. These procedures are especially useful for preparation and purification of post labeling enzymes: T4 polynucleotide kinase, T4 RNA ligase and of gamma 32P] ATP. We developed a test tube method for 5' 32P] pCp preparation followed by tRNA labeling with T4 RNA ligase. The parameters for optimal labeling were determined. Labeling of 3.10(6) to 5.10(6) Cerenkov CPM per microgram tRNA are currently obtained.