Strategies for Rescuing Plasmid DNA from Yeast Two-Hybrid Colonies
Once a yeast colony has been identified as containing a target plasmid insert of interest (as determined by growth on His− medium and a positive [blue] β-galactosidase assay; see Chapter 6), it becomes necessary to isolate the correct insert-containing plasmid. Isolating the plasmid DNA from yeast is not a trivial task, for several reasons. First, there is always contamination of the plasmid DNA by yeast genomic DNA since the isolation method breaks the yeast chromosomes. Second, most plasmids used tend to be large (>6 kb) and have a low copy number (∼50/cell), frequently resulting in low plasmid yields. And, finally, unlike bacteria, yeast are capable of replicating more than one plasmid at a time, making it difficult to identify the one containing the relevant insert. Thus, multiple steps are necessary to isolate the single insert-containing plasmid responsible for the interaction and activation and then to prepare it for analysis.
KeywordsVortex Proline Tryptophan Leucine Isopropanol
- 1.Clontech Laboratories. (1997) Clontech Yeast Protocols Handbook, protocol no. PT3024-1, version no. PR7X265.Google Scholar
- 2.Stratagene. (1997) HybriZAP®-2.1 two-hybrid cDNA gigapack cloning kit and HybriZAP®-2.1 two-hybrid cDNA synthesis kit Instruction Manual, manual no. 235612-12, revision no. 0870010.Google Scholar
- 5.Seidman, C. E., Struhl, K., Sheen, J., and Jessen, T. (1997) Introduction of plasmid DNA into cells, in Current Protocols in Molecular Biology John Wiley & Sons, New York, pp. 1.8.1–1.8.10.Google Scholar
- 6.Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.Google Scholar
- 8.Cowell, I. Yeast two-hybrid library screening. in Methods in Molecular Biology, vol. 69: cDNA Library Protocols (Cowell, I. G. and Austin, C. A., eds.), Humana, Totowa, NJ, pp. 185–202.Google Scholar