Please cite as: CSH Protocols; 2006; doi:10.1101/pdb.prot4389

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Simple, Reliable Steps for DNA Fragment Isolation and Purification

Andras Nagy, Marina Gertsenstein, Kristina Vintersten, and Richard Behringer

This protocol was adapted from "Production of Transgenic Mice," Chapter 7 of Manipulating the Mouse Embryo, 3rd edition, by Andras Nagy, Marina Gertsenstein, Kristina Vintersten, and Richard Behringer. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2003.

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


INTRODUCTION

This protocol describes a simple procedure for DNA fragment isolation and purification. The resulting DNA fragment can then be used for microinjection to produce transgenic mice.


MATERIALS

Reagents

Bacterial culture containing plasmid with transgenic construct, prepared by standard methods

GENECLEAN Turbo salt (BIO 101)

GENECLEAN Turbo Wash solution (BIO 101)

caution Methylene blue solution, 0.1% in 0.5 M sodium acetate (pH 5.2)

recipe Microinjection buffer

Relevant restriction enzymes

recipe caution Tris-acetate/EDTA (TAE) buffer

Equipment

BioTrap electroelution device (Schleicher & Schuell, Bio-Rad)

Electrophoresis chamber suitable for the BioTrap device

GENECLEAN Turbo columns (BIO 101)

Microcentrifuge tubes

Qiagene Endo Free Plasmid kit

Scalpel blade, clean


METHOD

1. Prepare plasmid DNA . . . [Full Text of this Article]


TROUBLESHOOTING


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