Cite as: Cold Spring Harb. Protoc.; 2006; doi:10.1101/pdb.prot4393

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Purification of Yeast Artificial Chromosome (YAC) DNA with Filtration Units

Lluis Montoliu

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 method for purification of YAC DNA using ultrafiltration. This is the fastest method available, but care needs to be taken not to damage the DNA. The purified DNA can then be used for microinjection.


MATERIALS

Reagents

Agarose gel

recipe Agarose gel, LMP (NuSieve GTG, FMC) in 1X TAE

Agarose plugs (see Large-Scale Preparation of Agarose Plugs of Yeast DNA)

recipe Equilibration buffer for YAC DNA

recipe caution Ethidium bromide (EtBr) stock solution (10 mg/ml)

Gelase (Epicentre, or Agarase from NEB)

Lambda DNA multimers (NEB or Boehringer Mannheim)

recipe Microinjection buffer for BAC/YAC DNA

SeaPlaque GTG (FMC) LMP

recipe TAE buffer, 0.5X, 1X

Equipment

Gel . . . [Full Text of this Article]


METHOD


TROUBLESHOOTING


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