Sodium Chloride → 5'-Thymidylic acid

To 300 ug of a solution comprising 10 mM Tris-HCl (pH 7.5), 6 mM MgCl2 and 10 mM NaCl, there was added 400 ug 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 ug of the KpnI-cleaved DNA was added to 200 ul 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 pCDVl. About 100 ug of the poly (dT) chain tailed pCDVl DNA was recovered from the solution by ethanol precipitation following phenol-chloroform extraction. The DNA was added to 1.50 ul of a solution comprising 0 mM Tris-HCl (pH 7.5), 6 M 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 ug of the poly(dT) chain-tailed pCDVl was thus obtained. The DNA was dissolved in 500 ul of a solution comprising 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA. the solution was incubated at 65° C. for 5 minutes and then cooled with ice, and 50 ul of 5 M 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 he column and they were eluted with a solution comprising 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA to give 27 ug of the poly(dT) chain-tailed pCDVl (hereinafter referred to as vector primer).

1. additionwas added 400 ug 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 ug of the KpnI-cleaved DNA was added to 200 ul 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 pCDVl
9. customwas recovered from the solution by ethanol precipitation
10. extractionphenol-chloroform extraction
11. additionThe DNA was added to 1.50 ul 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

ORD record

ord-1d6b92954c274830acc43abce1af7c63

Source dataset

uspto-grants-1999_11

DOI

10.6084/m9.figshare.5104873.v1

Patent number

US05994518