Unit

UNIT 5.9 Construction of Bacterial Artificial Chromosome (BAC/PAC) Libraries

  1. Kazutoyo Osoegawa1,
  2. Pieter J. de Jong1,
  3. Eirik Frengen2,
  4. Panayiotis A. Ioannou3

Published Online: 1 AUG 2001

DOI: 10.1002/0471142727.mb0509s55

Current Protocols in Molecular Biology

Current Protocols in Molecular Biology

How to Cite

Osoegawa, K., de Jong, P. J., Frengen, E. and Ioannou, P. A. 2001. Construction of Bacterial Artificial Chromosome (BAC/PAC) Libraries. Current Protocols in Molecular Biology. 55:IV:5.9:5.9.1–5.9.33.

Author Information

  1. 1

    Children's Hospital Oakland Research Institute, Oakland, California

  2. 2

    The Biotechnology Centre of Oslo University of Oslo, Oslo, Norway

  3. 3

    The Murdoch Institute for Research into Birth Defects, Royal Children's Hospital, Melbourne, Australia

Publication History

  1. Published Online: 1 AUG 2001
  2. Published Print: JUL 2001

Abstract

Large-insert genomic libraries are necessary for physical mapping of large chromosomal regions, for isolation of complete genes, and for use as intermediates in DNA sequencing of entire genomes. Construction of BAC and PAC libraries is detailed in the unit, including preparation of PAC or BAC vector DNA for cloning by digestion with BamHI or EcoRI, dephosphorylation with alkaline phosphatase, and purification through pulsed-field gel electrophoresis (PFGE). For the preparation of high-molecular weight DNA for cloning, procedures for embedding total genomic DNA from lymphocytes or animal tissue cells are also provided. Other protocols detail partial digestion of genomic DNA with MboI or with a combination of EcoRI endonuclease and EcoRI methylase (including methods for optimizing the extent of digestion), and subsequent size fractionation by preparative PFGE. Finally, the isolation of BAC and PAC plasmid DNA for analyzing clones is also presented.