Preparation of BAC Libraries From Bacterial Genomes by In Vitro Packaging

  • Sangita Pal
  • Solida Mak
  • George M. Weinstock
Part of the Methods in Molecular Biology™ book series (MIMB, volume 255)

Abstract

Genomic libraries represent a powerful resource for genetic studies of bacteria. Large-insert libraries in bacterial artificial chromosome (BAC) vectors are particularly important not only for genome-sequencing projects, but for comparative and functional genomic studies once a complete sequence is known. Cloned DNA can be introduced into bacterial cells in two different ways. One way is to have cells take up naked DNA; this is known as transformation (1). The other way is to package the recombinant DNA inside bacteriophage particles in vitro using a phage such as λ. This process, known as in vitro packaging, allows DNA to be introduced by infection (2). Although transformation allows DNA of any size to be introduced, DNA uptake occurs at very low frequency. On the other hand, in vitro packaging limits the size of the DNA based on the phage head stability. However, in vitro package is the most efficient method for introducing large-insert clones.

Keywords

Sucrose Glycerol EDTA MgSO4 Isopropanol 

References

  1. 1.
    Zhu, H. and Dean, R. A. (1999) A novel method for increasing the transformation efficiency of Escherichia coli—application for bacterial artificial chromosome library construction. Nucleic Acid Res. 27, 910–911.PubMedCrossRefGoogle Scholar
  2. 2.
    Champness, W. and Snyder, L. (1997) Molecular Genetics of Bacteria. American Society of Microbiology.Google Scholar
  3. 3.
    Weinstock, G. M., Maurer, R., and Berget, P. B. (1990) Advanced Bacterial Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.Google Scholar
  4. 4.
    Limberger, J. R., Silvenski, L. L., Izard, J., and Samsonoff, A. W. (1999) Insertional inactivation of Treponema denticola tap1 results in a nonmotile mutant with elongated flagellar hooks. J. Bacteriol. 181(12), 3743–3750.PubMedGoogle Scholar
  5. 5.
    Murray, M. G. and Thompson, W. F. (1980) Rapid isolation of high molecular weight plant DNA. Nucleic Acid Res. 8, 4321–4325.PubMedCrossRefGoogle Scholar
  6. 6.
    Sambrook, J., Fritsch, E. F, and Maniatis, T (1989) Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.Google Scholar
  7. 7.
    Ausubel, F M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G, Smith, J. A., and Straw, K. (1989) Current Protocols in Molecular Biology. John Wiley, New York.Google Scholar
  8. 8.
    Murray, E. M, Singh, K. V., Ross, R. P., Heath, J. D., Dunny, G. M., and Weinstock, G M. (1993) Generation of restriction map of Enterococcus faecalis OG1 and investigation of growth requirements and regions encoding biosynthetic function. J. Bacteriol. 175(16), 216–5222.Google Scholar
  9. 9.
    Teng, F, Murray, E. B., and Weinstock, G M. (1998) Conjugational transfer of plasmid DNA from E. coli to Enterococcus: A method to make insertional mutation. Plasmid 39, 182–186.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2004

Authors and Affiliations

  • Sangita Pal
    • 1
    • 2
  • Solida Mak
    • 1
  • George M. Weinstock
    • 2
  1. 1.Department of Microbiology and Molecular GeneticsUniversity of Texas-Houston Medical SchoolHouston
  2. 2.Human Genome Sequencing CenterBaylor College of MedicineHouston

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