Single-step marker switching in Schizosaccharomyces pombe using a lithium acetate transformation protocol

Simon David Brown, Alexander Lorenz

Research output: Contribution to journalArticle

Abstract

The ability to utilize different selectable markers for tagging or mutating multiple genes in Schizosaccharomyces pombe is hampered by the historical use of only two selectable markers, ura4+ and kanMX6; the latter conferring resistance to the antibiotic G418 (geneticin). More markers have been described recently, but introducing these into yeast cells often requires strain construction from scratch. To overcome this problem we and other groups have created transformation cassettes with flanking homologies to ura4+ and kanMX6 which enable an efficient and time-saving way to exchange markers in existing mutated or tagged fission yeast strains.
Here, we present a protocol for single-step marker switching by lithium acetate transformation in fission yeast, Schizosaccharomyces pombe. In the following we describe how to swap the ura4+ marker to a kanMX6, natMX4, or hphMX4 marker, which provide resistance against the antibiotics G418, nourseothricin (clonNAT) or hygromycin B, respectively. We also detail how to exchange any of the MX markers for nutritional markers, such as arg3+, his3+, leu1+ and ura4+.
Original languageEnglish
Article numbere2075
Number of pages11
JournalBio-protocol
Volume6
Issue number24
DOIs
Publication statusPublished - 20 Dec 2016

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lithium
Schizosaccharomyces pombe
geneticin
acetates
hygromycin B
yeasts
genes
cells

Keywords

  • schizosaccharomyces pombe
  • selectable marker
  • marker switch
  • Li-Acetate transformation
  • gene tagging
  • gene deletion
  • genetic manipulation

Cite this

Single-step marker switching in Schizosaccharomyces pombe using a lithium acetate transformation protocol. / Brown, Simon David; Lorenz, Alexander.

In: Bio-protocol, Vol. 6, No. 24, e2075, 20.12.2016.

Research output: Contribution to journalArticle

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abstract = "The ability to utilize different selectable markers for tagging or mutating multiple genes in Schizosaccharomyces pombe is hampered by the historical use of only two selectable markers, ura4+ and kanMX6; the latter conferring resistance to the antibiotic G418 (geneticin). More markers have been described recently, but introducing these into yeast cells often requires strain construction from scratch. To overcome this problem we and other groups have created transformation cassettes with flanking homologies to ura4+ and kanMX6 which enable an efficient and time-saving way to exchange markers in existing mutated or tagged fission yeast strains.Here, we present a protocol for single-step marker switching by lithium acetate transformation in fission yeast, Schizosaccharomyces pombe. In the following we describe how to swap the ura4+ marker to a kanMX6, natMX4, or hphMX4 marker, which provide resistance against the antibiotics G418, nourseothricin (clonNAT) or hygromycin B, respectively. We also detail how to exchange any of the MX markers for nutritional markers, such as arg3+, his3+, leu1+ and ura4+.",
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