Protocol

The Hanahan Method for Preparation and Transformation of Competent E. coli: High-efficiency Transformation

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

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INTRODUCTION

This procedure generates competent cultures of E. coli that can be transformed at high frequencies (5 x 10⁸ transformed colonies/µg of superhelical plasmid DNA). IMPORTANT All steps in this protocol should be carried out aseptically.

MATERIALS

E. coli strain to be transformed (frozen stock)

The strain should be stored at -70°C in freezing medium.

DMSO

DnD solution

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 Plasmid DNA, Addition of Synthetic Linkers to Blunt-ended DNAand Ligating . . . [Full Text of this Article]