An improved procedure for utilizing terminal transferase to add homopolymers to the 3' termini of DNA.
Terminal deoxynucleotidyl transferase (E.C.2.7.7.3.1.) from calf thymus was used to add homopolymer tails to duplex DNA with 3' protruding, even, or 3' recessive ends. A gel electrophoresis method was employed to analyze the tail length and the percent of DNA with tails. In all the tailing reactions, dA, dT, and dC tails from CoCl2-containing buffer were longer than those from MnCl2 - or MgCl2 - containing buffers, whereas dG tails from MnCl2 -containing buffer were the longest. By varying the ratio of dNTP over DNA terminus and the concentration of terminal transferase, optimal conditions were found for adding dG or dC tails of 10-25 nucleotides in length and dA and dT tails of 20-40 nucleotides in length to duplex DNA with all types of 3' termini.
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D003001:Cloning, Molecular / Q000379:methods*; D004253:DNA Nucleotidylexotransferase; D004254:DNA Nucleotidyltransferases; D004262:DNA Restriction Enzymes; D004274:DNA, Recombinant / Q000138:chemical synthesis*; D003854:Deoxyribonucleotides; D066298:In Vitro Techniques
D004274:DNA, Recombinant; D003854:Deoxyribonucleotides; D004254:DNA Nucleotidyltransferases; D004253:DNA Nucleotidylexotransferase; D004262:DNA Restriction Enzymes