反应详情

To 300 μl of a solution comprising 10 mM Tris-HCl (pH 7.5), 6 mM MgCl2 and 10 mM NaCl, there was added 400 μg of pCDV1 [Okayama & Berg: Mol. Cell. Biol., 3, 280 (1983)] and, after further addition of 500 units of KpnI, the reaction was carried out at 37° C. for 6 hours, whereby the plasmid was cleaved at the KpnI site. The DNA recovered byphenol-chloroform extraction followed 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 TdT buffer and, after further addition of 81 units of TdT (P-L Biochemicals), the reaction was carried out at 37° C. for 11 minutes, whereby a poly(dT) chain (about 67 dT residues) was added to each 3' end of the KpnI cleavage site of pCDV1. About 100 μg of the poly(dT) chain-tailed pCDV1 DNA was recovered from the solution by ethanol precipitation following phenol-chloroform extraction. The DNA was added to 150 μl of a solution comprising 10 mM Tris-HCl (pH 7.5), 6 mM MgCl2 and 100 mM NaCl and, after further addition of 360 units of EcoRI, the reaction was carried out at 37° C. for 2 hours. The reaction mixture was treated by the LGT method, and a DNA fragment of about 3.1 kb was recovered. About 60 μg of the poly(dT) chaintailed pCDV1 was thus obtained. The DNA was dissolved in 500 μl of a solution comprising 10 mM Tris-CHl (pH 8.0) and 1mM 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. Molecules having a sufficient poly(dT) chain length were adsorbed on the column and they were eluted with a solution comprising 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA to give 27 μg of the poly(dT) chain-tailed pCDV1 (hereinafter referred to as vector primer).

1. additionwas added 400 μg of pCDV1 [Okayama & Berg: Mol. Cell. Biol., 3, 280 (1983)]
2. customThe DNA recovered
3. extractionbyphenol-chloroform extraction
4. customfollowed by ethanol precipitation
5. additionAbout 200 μg of the KpnI-cleaved DNA was added to 200 μl of a solution
6. customprepared
7. waitthe reaction was carried out at 37° C. for 11 minutes
8. additionwhereby a poly(dT) chain (about 67 dT residues) was added to each 3' end of the KpnI cleavage site of pCDV1
9. customAbout 100 μg of the poly(dT) chain-tailed pCDV1 DNA was recovered from the solution by ethanol precipitation
10. extractionphenol-chloroform extraction
11. additionThe DNA was added to 150 μl of a solution
12. additionafter further addition of 360 units of EcoRI
13. waitthe reaction was carried out at 37° C. for 2 hours
14. additionThe reaction mixture was treated by the LGT method
15. customa DNA fragment of about 3.1 kb was recovered
16. customwas thus obtained
17. waitthe solution was incubated at 65° C. for 5 minutes
18. additionwas added
19. washwere eluted with a solution