Please cite as: CSH Protocols; 2006; doi:10.1101/pdb.prot3925

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Dephosphorylation of Plasmid DNA

Joseph Sambrook and David W. Russell

This protocol was adapted from Molecular Cloning, 3rd edition, by Joseph Sambrook and David W. Russell. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2001

The first 15% of the full text of this article appears below.


INTRODUCTION

During ligation in vitro, T4 DNA ligase will catalyze the formation of a phosphodiester bond between adjacent nucleotides only if one nucleotide carries a 5'-phosphate residue and the other carries a 3'-hydroxyl terminus. Recircularization of vector DNA can therefore be minimized by removing the 5'-phosphate residues from both termini of the linear, double-stranded plasmid DNA with alkaline phosphatase.


MATERIALS

Calf intestinal alkaline phosphatase (CIP)

recipe EDTA (0.5 M, pH 8.0)

recipe EGTA (0.5 M, pH 8.0)

Ethanol

Optional, please see Step 5.

recipe caution Phenol

caution Phenol:chloroform . . . [Full Text of this Article]


METHOD


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