Sodium Chloride → 5'-Thymidylic acid

Participants

Reaction Steps

Thus, 400 μg of pCDVl [Okayama & Berg: Mol. Cell. Biol., 3, 280 (1983)] was added to 300 μl of a solution containing 10 mM Tris-HCl (pH 7.5), 6 mM MgCl2 and 10 mM NaCl and, after further addition of 500 units of KpnI, the plasmid was cleaved at its KpnI site by 6 hours of reaction at 37° C. After phenol-chloroform extraction, DNA was recovered by ethanol precipitation. About 200 μg of the KpnI-cleaved DNA was added to 200 μl of a solution prepared by adding dTTP in a concentration of 0.25 mM to a buffer (hereinafter, TdT buffer) containing 40 mM sodium cacodylate, 30 mM Tris-HCl (pH 6.8). 1 mM CaCl2 and 0.1 mM dithiothreitol (hereinafter, DTT). After further addition of 81 units of terminal deoxynucleotidyl transferase (hereinafter, TdT) (P-L Biochemicals), the mixture was incubated at 37° C. for 11 minutes, whereby an approximately 67-mer poly(dT) chain was added to the KpnI cleavage site 3' terminus of pCDVl. Ethanol precipitation from the solution following phenol-chloroform extraction gave about 100 μg of poly(dT) chain-added pCDV1 DNA. This DNA was added to 150 μl of 10 mM Tris-HCl (pH 7.5) containing 6 mM MgCl2 and 100 mM NaCl and, after further addition of 360 units of EcoRI, the mixture was incubated at 37° C. for 2 hours. After treatment of the reaction mixture by the LGT method, a DNA fragment, 3.1 kb in length, was recovered. Thus was obtained about 60 μg of poly(dT) chain-added pCDV1. This DNA was dissolved in 500 μl of 10 mM Tris-HCl (pH 8.0) containing 1 mM EDTA, the solution was incubated at 65° C. for 5 minutes and then cooled with ice, and 50 μl of 5M NaCl was added. The mixture was subjected to oligo(dA)-cellulose column (Collaborative Research) chromatography. Fragments sufficient in (dT) chain length were adsorbed on the column. They were eluted with 10 mM Tris-HCl (pH 8.0) containing 1 mM EDTA to give 27 μg of poly(dT) chain-added pCDV1 (hereinafter briefly referred to as "vector primer").

Provenance & Evidence