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 was recovered by phenol-chloroform extraction and ethanol pre-cipitation. 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 abbreviated as TdT buffer) comprising 40 mM sodium cacodylate, 30 mM Tris-HCl (pH 6.8), 1 mM CaCl2 and 0.1 mM dithiothreitol (hereinafter abbreviated as DTT) and, after further addition of 81 units of terminal deoxy-nucleotidyl transferase (hereinafter abbreviated as TdT) (P-L Biochemicals), the reaction was carried out at 37° C. for 11 minutes, whereby a poly(dT) chain comprising about 67 dT residues was added to each KpnI cleavage site 3' end of pCDV1. About 100 μg of the poly(dT) chain-added pCDV1 DNA was recovered from the above reaction mixture by phenol-chloroform extraction and ethanol precipitation. The DNA was added to 150 μl of a buffer comprising 10 mM Tris-HCl (pH 7.5), 6 mM MgCl2 and 100 mM NaCl, 360 units of EcoRI was further added, and 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. Thus was obtained about 60 μg of the poly(dT) chain-tailed pCDV1. The DNA was dissolved in 500 μl 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 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. Thus was obtained 27 pg of the poly(dT) chain-tailed pCDV1 (hereinafter abbreviated as vector primer).

1. additionwas added 400 μg of pCDV1 [Okayama & Berg: Mol. Cell. Biol., 3, 280 (1983)]
2. additionafter further addition of,500 units of KpnI
3. customThe DNA was recovered by phenol-chloroform extraction and ethanol pre-cipitation
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. additionwas added to each KpnI cleavage site 3' end of pCDV1
8. customAbout 100 μg of the poly(dT) chain-added pCDV1 DNA was recovered from the above reaction mixture by phenol-chloroform extraction and ethanol precipitation
9. additionThe DNA was added to 150 μl of
10. additionwas further added
11. waitthe reaction was carried out at 37° C. for 2 hours
12. additionThe reaction mixture was treated by the LGT method
13. customa DNA fragment of about 3.1 kb was recovered

ORDレコード

ord-9ef1d031d1734bd5ad257b75c38b44b0

出典データセット

uspto-grants-1998_08

DOI

10.6084/m9.figshare.5104873.v1

特許番号

US05795968