Construction of Small‐Insert Libraries from Genomic DNA

Thomas J. Hudson1

1 Whitehead Institute, Cambridge, Massachusetts
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 2.1
DOI:  10.1002/0471142905.hg0201s00
Online Posting Date:  May, 2001
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Abstract

Construction of small‐insert genomic libraries involves ligation of a size‐selected insert to vector DNA. In this unit, vector DNA is prepared by digestion with a restriction endonuclease followed by phosphatase treatment to dephosphorylate the vector ends. Insert DNA is prepared by complete digestion of DNA with a compatible 4‐base‐cutting restriction endonuclease (e.g., AluI, HaeIII, RsaI, or Sau3A), as such frequently cutting endonucleases give the highest fragment number in the desired range. Alternatively, fragments may be generated by sonication of genomic DNA. Once the fragments have been generated, they are size selected on an agarose gel. The portion of the gel containing DNA of the desired size is excised and the DNA released from the agarose by digestion with b‐agarase. Insert and vector DNAs are then ligated with T4 DNA ligase and used to transform competent Escherichia coli cells to create a small‐insert library.

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1:

  MaterialsFor recipes, see in this unit (or cross‐referenced unit); for common stock solutions, see appendix 2D ; for suppliers, see suppliers appendix.
  • Plasmid or phagemid vector DNA
  • Selected blunt‐ or cohesive‐end‐generating restriction enzyme(s) and appropriate 10× buffer(s)
  • 10 mg/ml BSA
  • 0.8% agarose minigel
  • 0.5% µg/ml ethidium bromide ( appendix 2D)
  • 10×, 6×, and 1× gel loading buffer ( appendix 2D)
  • 10,000 U/ml calf intestine phosphatase (CIP) and 10× buffer
  • 0.5 M EDTA, pH 8.0
  • Buffered phenol ( appendix 3B)
  • 25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol ( appendix 3C)
  • Chloroform ( appendix 3C)
  • Purified genomic DNA ( appendix 3B)
  • DNA molecular size markers: HaeIII‐digested φX‐174 DNA, ligase inhibitor–free
  • 1000 U/ml β‐agarase and 10× buffer (New England Biolabs)
  • 3 M sodium acetate, pH 5.5
  • TE buffer
  • 10× T4 DNA ligase buffer ( appendix 2D) or recipe10× blunt‐end‐ligation buffer (see recipe)
  • 400,000 U/ml T4 DNA ligase
  • Competent Escherichia coli cells (CPMB UNITS & )
  • 100‐mm LB plates containing appropriate antibiotics and color‐selection reagents ( appendix 2D)
  • 65°, 40°, and 16°C water baths
  • 0.5‐ml and graduated 1.5‐ml microcentrifuge tubes
  • Additional reagents and equipment for agarose gel electrophoresis using minigels and large‐scale gels (unit 2.7), phenol extraction and ethanol and isopropanol precipitation ( appendix 3C), quantitation of DNA using fluorochrome Hoechst 33258 ( appendix 3D), and introduction of plasmid or phage DNA into cells (CPMB UNITS & )
CAUTION: Ethidium bromide, phenol, and chloroform are hazardous; see appendix 2A for safety guidelines.
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Figures

Videos

Literature Cited

   Johnson, D.H. 1990. Molecular cloning of DNA from specific chromosomal regions by microdissection and sequence‐independent amplification of DNA. Genomics 6:243‐251.
   New England Biolabs Catalog. 1992. New England Biolabs, Beverly, MA.
   Sambrook, J., Fritsch, E.F., and Maniatis, T. 1989. Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
   Young, B.D. 1990. Chromosome Analysis and Sorting. In Flow Cytometry (M.G. Ormerod, ed.) pp. 145‐159. Oxford University Press, Oxford.
Key Reference
   Hudson, T.J., Engelstein, M., Lee, M.K., Ho, E.C., Rubenfield, M.J., Adams, C.P., Housman, D.E., and Dracopoli, N.C. 1992. Isolation and chromosomal assignment of 100 highly informative human simple sequence repeat polymorphisms. Genomics 13:622‐629.
  Describes isolation of CA and tetranucleotide repeats from small‐insert genomic libraries using the approach described herein.
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