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Duplication and Transformation of the Schizosaccharomyces pombe Collection of Deletion Strains

  • Sudhir Kumar Rai
  • Angela Atwood-Moore
  • Henry L. LevinEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1721)

Abstract

We present an efficient and organized method of lithium acetate and polyethylene glycol-based transformation of plasmid DNA into the commercially available collection of Schizosaccharomyces pombe with single-gene deletions. We also describe how to prepare a duplicate collection of the deletion strains in order to preserve the longevity of the master set. These protocols are adapted to the 96-well format of the 3004 strains of the Version 2.0 Bioneer set but can also be used for later releases of the collection. This transformation method typically yields efficiencies in the range between 1.0 × 103 and 1.0 × 104 transformants per microgram of plasmid DNA. However, some deletion strains transformed with significantly lower efficiencies. We provide a list of these difficult-to-transform strains. Applications for this methodology include the transformation of the deletion set with plasmids necessary for genetic screens.

Key words

Tf1 Schizosaccharomyces pombe Fission yeast Lithium acetate Polyethylene glycol Transformation Commercial deletion library 

References

  1. 1.
    Wood V, Harris MA, McDowall MD, Rutherford K, Vaughan BW, Staines DM, Aslett M, Lock A, Bahler J, Kersey PJ, Oliver SG (2011) PomBase: a comprehensive online resource for fission yeast. Nucleic Acids Res 40(Database issue):D695–D699PubMedPubMedCentralGoogle Scholar
  2. 2.
    Wood V, Gwilliam R, Rajandream MA, Lyne M, Lyne R, Stewart A, Sgouros J, Peat N, Hayles J, Baker S, Basham D, Bowman S, Brooks K, Brown D, Brown S, Chillingworth T, Churcher C, Collins M, Connor R, Cronin A, Davis P, Feltwell T, Fraser A, Gentles S, Goble A, Hamlin N, Harris D, Hidalgo J, Hodgson G, Holroyd S, Hornsby T, Howarth S, Huckle EJ, Hunt S, Jagels K, James K, Jones L, Jones M, Leather S, McDonald S, McLean J, Mooney P, Moule S, Mungall K, Murphy L, Niblett D, Odell C, Oliver K, O'Neil S, Pearson D, Quail MA, Rabbinowitsch E, Rutherford K, Rutter S, Saunders D, Seeger K, Sharp S, Skelton J, Simmonds M, Squares R, Squares S, Stevens K, Taylor K, Taylor RG, Tivey A, Walsh S, Warren T, Whitehead S, Woodward J, Volckaert G, Aert R, Robben J, Grymonprez B, Weltjens I, Vanstreels E, Rieger M, Schäfer M, Müller-Auer S, Gabel C, Fuchs M, Düsterhöft A, Fritzc C, Holzer E, Moestl D, Hilbert H, Borzym K, Langer I, Beck A, Lehrach H, Reinhardt R, Pohl TM, Eger P, Zimmermann W, Wedler H, Wambutt R, Purnelle B, Goffeau A, Cadieu E, Dréano S, Gloux S, Lelaure V, Mottier S, Galibert F, Aves SJ, Xiang Z, Hunt C, Moore K, Hurst SM, Lucas M, Rochet M, Gaillardin C, Tallada VA, Garzon A, Thode G, Daga RR, Cruzado L, Jimenez J, Sánchez M, del Rey F, Benito J, Domínguez A, Revuelta JL, Moreno S, Armstrong J, Forsburg SL, Cerutti L, Lowe T, McCombie WR, Paulsen I, Potashkin J, Shpakovski GV, Ussery D, Barrell BG, Nurse P (2002) The genome sequence of Schizosaccharomyces pombe. Nature 415(6874):871–880CrossRefPubMedGoogle Scholar
  3. 3.
    Kim DU, Hayles J, Kim D, Wood V, Park HO, Won M, Yoo HS, Duhig T, Nam M, Palmer G, Han S, Jeffery L, Baek ST, Lee H, Shim YS, Lee M, Kim L, Heo KS, Noh EJ, Lee AR, Jang YJ, Chung KS, Choi SJ, Park JY, Park Y, Kim HM, Park SK, Park HJ, Kang EJ, Kim HB, Kang HS, Park HM, Kim K, Song K, Song KB, Nurse P, Hoe KL (2010) Analysis of a genome-wide set of gene deletions in the fission yeast Schizosaccharomyces pombe. Nat Biotechnol 28(6):617–623CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Rai SK, Sangesland M, Lee M Jr, Esnault C, Cui Y, Chatterjee AG, Levin HL. (2017) Host factors that promote retrotransposon integration are similar in distantly related eukaryotes. PLoS Genet. 13(12):e1006775. doi:  10.1371/journal.pgen.1006775. [Epub ahead of print] PMID: 29232693 [PubMed - as supplied by publisher]
  5. 5.
    Sangesland M, Atwood-Moore A, Rai SK, Levin HL (2016) Qualitative and quantitative assays of transposition and homologous recombination of the retrotransposon Tf1 in Schizosaccharomyces pombe. Methods Mol Biol 1400:117–130CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  • Sudhir Kumar Rai
    • 1
    • 2
  • Angela Atwood-Moore
    • 1
  • Henry L. Levin
    • 1
    Email author
  1. 1.Section on Eukaryotic Transposable Elements, Division of Molecular and Cellular Biology, The Eunice Kennedy Shriver National Institute of Child Health and Human DevelopmentNational Institutes of HealthBethesdaUSA
  2. 2.Division of Hematology/Oncology, Department of MedicineUniversity of FloridaGainesvilleUSA

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