Unit

UNIT 5.15 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 MAY 2001

DOI: 10.1002/0471142905.hg0515s21

Current Protocols in Human Genetics

Current Protocols in Human Genetics

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 Human Genetics. 21:5.15:5.15.1–5.15.33.

Author Information

  1. 1

    Roswell Park Cancer Institute, Buffalo, New York

  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 MAY 2001
  2. Published Print: MAY 1999

Abstract

This unit describes the construction of BAC and PAC libraries. Two vectors, pCYPAC2 and pPAC4 have been used for preparing PAC libraries, and a new BAC vector pBACe3.6 has been developed for construction of BAC libraries. A support protocol describes preparation of PAC or BAC vector DNA for cloning by digestion with BamHI or EcoRI, simultaneous dephosphorylation with alkaline phosphatase, and subsequent purification through pulsed-field gel electrophoresis (PFGE). For the preparation of high-molecular weight DNA for cloning, support protocols provide procedures for embedding total genomic DNA from lymphocytes or animal tissue cells, respectively, in InCert agarose. Another support protocol details the next steps for the genomic DNA: partial digestion with MboI or with a combination of EcoRI endonuclease and EcoRI methylase, and subsequent size fractionation by preparative PFGE. The final support protocol covers the isolation of BAC and PAC plasmid DNA for analyzing clones.