Rescuing YAC‐Insert Ends as E. coli Plasmids

Gary G. Hermanson1, Glen A. Evans2

1 Cytel Corporation, San Diego, California, 2 The Salk Institute, San Diego, California
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 5.8
DOI:  10.1002/0471142905.hg0508s00
Online Posting Date:  May, 2001
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Abstract

End clones from YACs (terminal fragments of the YAC‐insert DNA cloned into a plasmid vector) are essential ingredients for contig building using chromosome walking strategies, for fluorescent in situ hybridization experiments, and for generating repeat‐free probes for pulsed‐field gel electrophoresis or genetic linkage analysis. The describes a method for rescuing the CEN (centromere) ends of YACs constructed in the vector pYAC4. However, because this method relies on XhoI and SalI sites, which are relatively rare in the mammalian genome, it is not always possible to obtain a subclone in this manner. An alternate protocol presents a method utilizing integrative plasmid‐rescue vectors to facilitate the isolation of both ends of any YAC clone even in the absence of convenient restriction enzyme sites.

     
 
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Table of Contents

  • Basic Protocol 1: Plasmid Rescue by XhoI or XhoI + SalI Circularization
  • Alternate Protocol 1: Plasmid End Rescue Using URA‐Side Integrative Vector
  • Alternate Protocol 2: Plasmid End Rescue Using CEN‐Side Integrative Vector
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Plasmid Rescue by XhoI or XhoI + SalI Circularization

  Materials
    For recipes, see Reagents and Solutions in this unit (or cross‐referenced unit); for common stock solutions, see appendix 2D; for suppliers, see suppliers appendix.
  • Yeast culture containing a YAC clone
  • YPD medium (unit 5.5)
  • SalI and/or XhoI restriction endonucleases
  • 10× universal restriction enzyme buffer (see recipe)
  • 25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol ( appendix 3C)
  • Chloroform
  • TE buffer, pH 7.4
  • 10× T4 DNA ligase buffer ( appendix 2D)
  • 5 U/µl T4 DNA ligase (in cohesive‐end units)
  • Electroporation‐competent E. coli ( CPMB UNIT 1.8)
  • LB plates containing 50 µg/ml ampicillin ( appendix 2D)
  • 30°C shaking incubator
  • Additional reagents and equipment for preparing yeast genomic DNA from YAC‐bearing clones (unit 5.9), phenol extraction and ethanol precipitation of DNA ( appendix 3C), electroporation ( CPMB UNIT 1.8), plasmid DNA miniprep (unit 5.3 or CPMB UNIT 1.6), and agarose gel electrophoresis (unit 2.7)
CAUTION: Phenol and chloroform are hazardous; see appendix 2A for guidelines on handling, storage, and disposal.

Alternate Protocol 1: Plasmid End Rescue Using URA‐Side Integrative Vector

  Additional MaterialsFor recipes, see Reagents and Solutions in this unit (or cross‐referenced unit); for common stock solutions, see appendix 2D; for suppliers, see suppliers appendix .
  • Yeast containing the YAC clone of interest, made transformation competent by lithium acetate treatment (unit 5.7) or spheroplasting (unit 5.2)
  • pLUS vector (ATCC #77407 or Dr. Glen Evans, Salk Institute) linearized by cleavage with Sal I
  • SORB dropout plates (unit 5.2) without uracil or lysine (−Ura −Lys) and containing 10 mg/liter adenine
  • 10× PCR amplification buffer containing 15 mM MgCl 2 ( appendix 2D)
  • Dimethylsulfoxide (DMSO)
  • 2.5 mM 4dNTP mix ( appendix 2D)
  • 25 µM pLUS PCR primers (see recipe)
  • 2.5 U/µl Taq DNA polymerase
  • Restriction enzyme with recognition site in the pLUS polylinker: BamHI, KpnI, SacI, XbaI, or HindIII
  • LB plates containing 25 µg/ml kanamycin ( appendix 2D)
  • Additional reagents and equipment for yeast transformation (unit 5.2 or ), and isolation of yeast genomic DNA by spheroplasting (unit 5.9) or glass bead method ( CPMB UNIT 13.11)

Alternate Protocol 2: Plasmid End Rescue Using CEN‐Side Integrative Vector

  Additional MaterialsFor recipes, see Reagents and Solutions in this unit (or cross‐referenced unit); for common stock solutions, see appendix 2D; for suppliers, see suppliers appendix .
  • pICL vector (ATCC #77408 or Dr. Glen Evans, Salk Institute) linearized by cleavage with Sca I
  • pICL PCR primers (see recipe)
  • PstI restriction endonuclease
  • Restriction enzyme with recognition site in the pICL polylinker: BamHI, Kpn I, SphI, or SacI
Follow the procedure in the previous alternate protocol, with the following modifications at the indicated steps:
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Figures

Videos

Literature Cited

Literature Cited
   Burke, D.T., Carle, G.F., and Olson, M.V. 1987. Cloning of large segments of exogenous DNA into yeast by means of artificial chromosome vectors. Science 236:806‐812.
   Garza, D., Ajioka, J.W., Burke, D.T., and Hartl, D.L. 1989. Mapping the Drosophila genome with yeast artificial chromosomes. Science 246:641‐646.
   McCormick, M.K., Shero, J.H., Cheung, M.C., Kan, Y.W., Hieter, P.A., and Antonarakis, S.E. 1989. Construction of human chromosome 21–specific yeast artificial chromosomes. Proc. Natl. Acad. Sci. U.S.A. 86:9991‐9995.
Key Reference
   Hermanson, G.G., Hoekstra, M., McElligott, D.L., and Evans, G.A. 1991. Rescue of end fragments of yeast artificial chromosomes by homologous recombination in yeast. Nucl. Acids Res. 19:4943‐4948.
  Describes CEN‐side and URA‐side integrative plasmid rescue.
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