Making temperature-sensitive mutants

Shay Ben-Aroya, Xuewen Pan, Jef D. Boeke, Philip Hieter

Research output: Contribution to journalArticlepeer-review


The study of temperature-sensitive (Ts) mutant phenotypes is fundamental to gene identification and for dissecting essential gene function. In this chapter, we describe two "shuffling" methods for producing Ts mutants using a combination of PCR, in vivo recombination, and transformation of diploid strains heterozygous for a knockout of the desired mutation. The main difference between the two methods is the type of strain produced. In the "plasmid" version, the product is a knockout mutant carrying a centromeric plasmid carrying the Ts mutant. In the "chromosomal" version, The Ts alleles are integrated directly into the endogenous locus, albeit not in an entirely native configuration. Both variations have their strengths and weaknesses, which are discussed here.

Original languageEnglish (US)
Pages (from-to)181-204
Number of pages24
JournalMethods in enzymology
Issue numberC
StatePublished - 2010

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology


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