The isolation of enzymically modified tRNA's from enzyme reaction mixtures is usually achieved by one of two general met,hods. Amino-acyl synthetase reactions are easily assayed by filt'er paper techniques (1) in which t,he tRNA is precipitated on filters of materials such as cellulose ester or glass fiber. After drying, the filters can be counted by several techniques. The limitations of this technique include unknown counting efficiency and loss of the sample. In cases in which recovery of the tRNA is desired the usual procedure involves phenol extraction of the tRNA followed by selective precipitation with sa1t.s and/or organic solvents. We wish to report a technique for the rapid isolat,ion of tRNA from reaction mixtures which leads to the quantitative recovery of tRNA essentially free of protein. The assay depends on the selective adsorption to and elution from microgranular DEAE in the presence of low and high salt, respectively.
Methods
Whatman DE32 microgranular DEAE-cellulose was obtained from Reeve Angel Company. It is equilibrated in buffer (10 gm dry DEAE in 100 ml 0.1 .M Tris-HCl, pH 7.5/0.1 M NaCl or 0.1 M sodium acetate, pH 5.0/0.1 M NaCl) and just prior to use the suspension is well mixed to give a uniform preparation.
Unfractionated Escherichia coli B tRNA, lots 7001 and V2040, was obtained from Schwarz BioResearch. 3H-Phenylalanine (specific activity 406 mCi/mmole) was obtained from Amersham/Searle Corp. and diluted to 50 mCi/mmole with unlabeled phenylalanine from Schwarz Bio-Research. Crystalline BSA was obtained from British Drug Houses. Carrier RNA was prepared from unfractionated yeast RNA (British Drug Houses) by the met'hod of Singer and Tolbert (2).