A Rapid PCR-Based Colony Screening Protocol for Cloned Inserts

  • Michael K. Trower
Protocol
Part of the Methods in Molecular Biology™ book series (MIMB, volume 58)

Abstract

Following transformation of a ligation reaction into competent E. coli cells, successful subclones are conventionally identified by two methods. The first involves preparing “mini-prep” plasmid DNA from a number of colonies and then identifying the desired recombinant plasmid on the basis of either its unique restriction enzyme digest pattern or by direct DNA sequencing. The second, often used when large numbers of putative recombinants are involved, is by colony hybridization with a labeled probe. In this chapter an alternative PCR-based method for direct screening of transformants is described that is both facile and rapid, completely circumventing time-consuming DNA plasmid preparations. In its simplest form described below, transformed colonies are directly tooth-picked into a small volume of PCR reaction mix that includes primers that flank the cloning site. This is usually achieved with Universal primers from the vector polylinker (1). Following in vitro amplification, aliquots of each reaction are analyzed by agarose gel electrophoresis, which reveals both the presence and size of cloned inserts.

Keywords

EDTA Agarose Electrophoresis MgCl2 dNTP 

References

  1. 1.
    Güssow, D. and Clackson, T. (1989) Direct clone characterization from plaques and colonies by the polymerase chain reaction. Nucleic Acids Res. 17, 4000.PubMedCrossRefGoogle Scholar
  2. 2.
    Trower, M. K. (1992) A raped PCR dependent microtitre plate screening method for DNA sequences altered by site-directed mutagenesis. DNA Sequence—J. DNA Sequenc. Map. 3, 233–235.CrossRefGoogle Scholar
  3. 3.
    Trower, M. K., Burt, D., Purvis, I. J., Dykes, C. W., and Christodoulou, C. (1995) Fluorescent dye-primer cycle sequencing using unpurzfied PCR products; development of a protocol amenable to high-throughput DNA sequencing. Nucl. Acids Res. 23, 2348,2349.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 1996

Authors and Affiliations

  • Michael K. Trower
    • 1
  1. 1.Molecular Pathology DepartmentGlaxo Research and Development Inc.GreenfordUK

Personalised recommendations