Please cite as: CSH Protocols; 2006; doi:10.1101/pdb.prot3921

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Blunt-ended Cloning into Plasmid Vectors

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

Target DNA is ligated to a blunt-ended plasmid DNA, and the products of the ligation reaction are used to transform competent E. coli. The maximum number of "correct" clones can generally be obtained from ligation reactions containing equimolar amounts of plasmid and target DNAs, with the total DNA concentration being <100 µg/ml. Blunt-end ligation catalyzed by bacteriophage T4 DNA ligase is suppressed by high concentrations (5 mM) of ATP and polyamines such as spermidine.


MATERIALS

recipe ATP . . . [Full Text of this Article]


METHOD


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