Phenol → 5'-Thymidylic acid

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.

1. extractionThe mixture is extracted with equal volume of diethyl ether
2. additionfollowed by the addition of ethanol
3. customto form a precipitate
4. additionInto the mixture is added 17 units of λ-exonuclease
5. customto form a precipitate
6. additionThe precipitate (cleaved pBR322) is added to a 300 μl reaction mixture
7. waitfollowed by incubation at 37° C. for 20 minutes
8. extractionThen the mixture is extracted with phenolchloform
9. additionfollowed by the addition of ethanol
10. customto recover plasmid

ORD record

ord-77424235a43e43d2a387cced32b2bfe7

Source dataset

uspto-grants-1984_07

DOI

10.6084/m9.figshare.5104873.v1

Patent number

US04457867