Sodium Chloride → 5'-Thymidylic acid

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 by phenol-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) chain-tailed pCDV1 was thus obtained. The DNA was dissolved in and 1 mM EDTA, the solution was incubated at 65° C. for 5 minutes and then cooled with ice, and 50 μl 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 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 by phenol-chloroform extraction
3. customfollowed by ethanol precipitation
4. additionAbout 200 μg of the KpnI-cleaved DNA was added to 200 μl of a solution
5. customprepared
6. waitthe reaction was carried out at 37° C. for 11 minutes
7. additionwhereby a poly(dT) chain (about 67 dT residues) was added to each 3' end of the KpnI cleavage site of pCDV1
8. customwas recovered from the solution by ethanol precipitation
9. extractionphenol-chloroform extraction
10. additionThe DNA was added to 150 μl of a solution
11. additionafter further addition of 360 units of EcoRI
12. waitthe reaction was carried out at 37° C. for 2 hours
13. additionThe reaction mixture was treated by the LGT method
14. customa DNA fragment of about 3.1 kb was recovered

ORD record

ord-3c18e21f25174b87a4db238ed5af1c90

Source dataset

uspto-grants-1993_03

DOI

10.6084/m9.figshare.5104873.v1

Patent number

US05194592