Please cite as: CSH Protocols; 2006; doi:10.1101/pdb.prot3811

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protocolProtocol

Preparation of Uracil-containing Single-stranded Bacteriophage M13 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 100 words of the full text of this article appear below.


INTRODUCTION

Single-stranded templates of bacteriophage M13 DNA containing 20-30 residues of uracil in place of thymine are generated during growth of the bacteriophage in an F' strain of E. coli carrying mutations in the ung and dut genes. This DNA is used as a template in the Kunkel method of oligonucleotide-directed mutagenesis (Oligonucleotide-directed Mutagenesis of Single-stranded DNA).


MATERIALS

recipe 2x YT medium

recipe 2x YT medium containing 0.25 µg/ml uridine

E. coli strain CJ236 (dut- ung- F')

E. coli strain TG1, JM109, or equivalent

Please see Step 6.

Ethanol

Optional, please see Step 5.

Marker DNA

As a reference, use single-stranded . . . [Full Text of this Article]


METHOD


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