반응 #1549156

반응 방정식

sodium cacodylate

MgCl2

dTTP

phenol

Tris-HCl

MgCl2

NaCl

2-mercaptoethanol

sodium glycinate

MgCl2

→

dTMP

용매

—

mixture

ethanol

반응 조건

후처리

1. 1
   추출The mixture is extracted with equal volume of diethyl ether
2. 2
   workup.ADDITIONfollowed by the addition of ethanol
3. 3
   기타to form a precipitate
4. 4
   workup.ADDITIONInto the mixture is added 17 units of λ-exonuclease
5. 5
   기타to form a precipitate
6. 6
   workup.ADDITIONThe precipitate (cleaved pBR322) is added to a 300 μl reaction mixture
7. 7
   workup.WAITfollowed by incubation at 37° C. for 20 minutes
8. 8
   추출Then the mixture is extracted with phenolchloform
9. 9
   workup.ADDITIONfollowed by the addition of ethanol
10. 10
    기타to recover plasmid

실험 절차

20 μg of plasmid pBR322 and 10 units of EcoRI (manufactured and sold by Bethesda Research Laboratories, Inc., U.S.A.) are incubated at 37° C. for 2 hours in a 150 μl mixture containing 10 mM Tris-HCl (pH 7.5), 6 mM MgCl2, 50 mM NaCl, 6 mM 2-mercaptoethanol and 200 μg/μl BSA. The mixture is extracted with equal volume of diethyl ether, followed by the addition of ethanol to form a precipitate. Then, the precipitate is dissolved in a 100 μl mixture containing 0.1 mM sodium glycinate (pH 9.5), 5 mM MgCl2 and 50 μg/μl BSA. Into the mixture is added 17 units of λ-exonuclease, followed by incubation at 37° C. for one hour. Then, the mixture is subjected to phenol treatment, followed by the addition of ethanol to form a precipitate. The precipitate (cleaved pBR322) is added to a 300 μl reaction mixture containing 100 mM sodium cacodylate (pH 7.2), 10 mM Na2HPO4, 5 mM MgCl2, 1 mM dTTP, 50 μg/μl BSA and 3-6 units/μg (DNA) TdT, followed by incubation at 37° C. for 20 minutes. Then the mixture is extracted with phenolchloform, followed by the addition of ethanol to recover plasmid.

출처