Réaction #336597

Équation de réaction

Tris-HCl

DTT

NaCl

sodium cacodylate

dTTP

dithiothreitol

MgCl2

CaCl2

Tris-HCl

→

poly(dT)

Solvants

Conditions de réaction

Traitement

1. 1
   workup.ADDITIONwas added 400 μg of pCDV1 [Okayama & Berg: Mol. Cell. Biol., 3, 280 (1983)]
2. 2
   AutreThe DNA was recovered by phenol-chloroform extraction and ethanol precipitation
3. 3
   workup.ADDITIONAbout 200 μg of the KpnI-cleaved DNA was added to 200 μl of a solution
4. 4
   Autreprepared
5. 5
   workup.WAITthe reaction was carried out at 37° C. for 11 minutes
6. 6
   workup.ADDITIONwhereby a poly(dT}chain comprising about 67 dT residues was added to each KpnI cleavage site 3' end of pCDV1
7. 7
   AutreAbout 100 μg of the poly(dT) chain-added pCDV1 DNA was recovered from the above reaction mixture by phenol-chloroform extraction and ethanol precipitation
8. 8
   workup.ADDITIONThe DNA was added to 150 μl of
9. 9
   workup.ADDITIONwas further added
10. 10
    workup.WAITthe reaction was carried out at 37° C. for 2 hours
11. 11
    workup.ADDITIONThe reaction mixture was treated by the LGT method
12. 12
    Autrea DNA fragment of about 3.1 kb was recovered

Mode opératoire

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 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 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 deoxynucleotidyl 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 μg of the poly(dT) chain-tailed pCDV1 (hereinafter abbreviated as vector primer).

Source