Protocol

The Inoue Method for Preparation and Transformation of Competent E. Coli: "Ultra-Competent" Cells

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 protocol reproducibly generates competent cultures of E. coli that yield 1 x 10⁸ to 3 x 10⁸ transformed colonies/µg of plasmid DNA. The protocol works optimally when the bacterial culture is grown at 18°C. If a suitable incubator is not available, a standard bacterial shaker can be set up in a 4°C cold room and regulated to 18°C.

MATERIALS

DMSO

Inoue transformation buffer (please see Step 1)

Chilled to 0°C before use.

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 . . . [Full Text of this Article]