Please cite as: CSH Protocols; 2006; doi:10.1101/pdb.prot3932

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Preparation and Transformation of Competent E. coli Using Calcium Chloride

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 100 words of the full text of this article appear below.


INTRODUCTION

This protocol, developed approx. 30 years ago, is used to prepare batches of competent bacteria that yield 5 x 106 to 2 x 107 transformed colonies/µg of supercoiled plasmid DNA.


MATERIALS

recipe caution CaCl2•2H2O (1 M)

Standard transformation buffer (TFB) (please see The Hanahan Method for Preparation and Transformation of Competent E. coli: High-efficiency Transformation, Step 1) may be used in Step 8.

recipe MgCl2-CaCl2 solution, ice cold

Plasmid DNA (recombinant plasmid)

Construct using one of the methods described in Directional Cloning into Plasmid Vectors, Attaching Adaptors to Protruding Termini, Blunt-ended Cloning into Plasmid Vectors, Dephosphorylation of . . . [Full Text of this Article]


METHOD


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