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Construction of BAC Libraries from Flow-Sorted Chromosomes

  • Jan ŠafářEmail author
  • Hana Šimková
  • Jaroslav Doležel
Protocol
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Part of the Methods in Molecular Biology book series (MIMB, volume 1429)

Abstract

Cloned DNA libraries in bacterial artificial chromosome (BAC) are the most widely used form of large-insert DNA libraries. BAC libraries are typically represented by ordered clones derived from genomic DNA of a particular organism. In the case of large eukaryotic genomes, whole-genome libraries consist of a hundred thousand to a million clones, which make their handling and screening a daunting task. The labor and cost of working with whole-genome libraries can be greatly reduced by constructing a library derived from a smaller part of the genome. Here we describe construction of BAC libraries from mitotic chromosomes purified by flow cytometric sorting. Chromosome-specific BAC libraries facilitate positional gene cloning, physical mapping, and sequencing in complex plant genomes.

Key words

BAC library BAC vector Chromosomes DNA cloning High molecular weight (HMW) DNA Pulse-field gel electrophoresis (PFGE) Clone 

Notes

Acknowledgment

This work was supported by grant LO1204 from the National Program of Sustainability I. We also thank Dr. Zbyněk Milec for creating figures and critical reading of the manuscript.

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Jan Šafář
    • 1
    Email author
  • Hana Šimková
    • 1
  • Jaroslav Doležel
    • 1
  1. 1.Centre of the Region Haná for Biotechnological and Agricultural ResearchInstitute of Experimental BotanyOlomoucCzech Republic

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