Production of a Subtracted cDNA Library

Lloyd B. Klickstein1

1 Brigham and Women's Hospital, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 25B.1
DOI:  10.1002/0471142727.mb25b01s55
Online Posting Date:  August, 2001
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Abstract

For some experiments, a complete cDNA library is unnecessary and instead, a subtracted cDNA library is useful. A subtracted cDNA library contains cDNA clones corresponding to mRNAs present in one cell or tissue type and not present in a second type. This cDNA library is used to isolate a set of cDNA clones corresponding to a class of mRNAs, or to aid in the isolation of a cDNA clone corresponding to a particular mRNA where the screening procedure for the cDNA clone is laborious because a specific DNA or antibody probe is unavailable. Since relatively few recombinants are obtained after subtraction, this protocol is for a cDNA library constructed in the λ>10 vector or its equivalent, which allows a high cloning efficiency and permits elimination of nonrecombinants; however, the protocol can be used to produce subtracted cDNA libraries in any vector system.

     
 
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Table of Contents

  • Basic Protocol 1: Production of a Subtracted Library
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Production of a Subtracted Library

  Materials
  • [+] and [−] cDNA libraries (ATCC or Stratagene)
  • TE buffer ( appendix 22)
  • EcoRI and 10× EcoRI buffer (unit 3.1)
  • 0.5 M EDTA, pH 8.0 ( appendix 22)
  • 10% sucrose solution (unit 5.3)
  • 1.5% and 2% agarose gels (unit 2.5)
  • TBE buffer ( appendix 22)
  • 95% and 70% ethanol
  • S1 nuclease (Sigma; unit 3.12) and 10× S1 nuclease buffer (unit 3.4)
  • 25:24:1 phenol/chloroform/isoamyl alcohol (unit 2.1)
  • 3 M sodium acetate, pH 5.2 ( appendix 22)
  • AluI and 10× AluI buffer (unit 3.1)
  • RsaI (unit 3.1)
  • Deionized formamide (Fluka, IBI, or American Bioanalytical)
  • 20× SSC ( appendix 22)
  • recipe1 M NaPO 4, pH 7.0 (see recipe)
  • 10% sodium dodecyl sulfate (SDS)
  • 10 mg/ml yeast tRNA
  • 24:1 chloroform/isoamyl alcohol
  • Phosphatased λgt10 arms (Stratagene)
  • 10× T4 DNA ligase buffer (unit 3.4)
  • T4 DNA ligase (measured in cohesive‐end units; New England Biolabs; unit 3.14)
  • E. coliC600hflA (Table 97.80.4711)
  • λ phage packaging extracts (Stratagene)
  • Suspension medium (SM; unit 1.11)
  • SW‐28 rotor and 38‐ml centrifuge tubes (Beckman) or equivalent
  • 0.4‐ml microcentrifuge tube
  • Additional reagents and equipment for construction of recombinant DNA libraries (units 5.5 & 5.6), large‐scale DNA preps from plasmids (unit 1.7) or phage (unit 1.13), sucrose gradients (unit 5.3), agarose gel electrophoresis (unit 2.5), production and growth/maintenance of λ phage libraries (units 5.8, 25.2, and 1.9 1.3), plating and titering libraries (units 6.1 & 6.2), hybridization (unit 6.3), and radiolabeling probes (unit 3.4)
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Figures

Videos

Literature Cited

Literature Cited
   Hedrick, S.M., Cohen, D.I., Nielsen, E.A., and Davis, M.M. 1984. Isolation of cDNA clones encoding T cell–specific membrane‐associated proteins. Nature (Lond.) 308:149‐153.
   Lamar, E.E. and Palmer, E. 1984. Y‐encoded, species‐specific DNA in mice: Evidence that the Y chromosome exists in two polymorphic forms in inbred strains. Cell 37:171‐177.
   Tedder, T.F., Strueli, M., Schlossman, S.F., and Saito, H. 1988. Isolation and structure of a cDNA encoding the B1 (CD20) cell‐surface antigen of human B lymphocytes. Proc. Natl. Acad. Sci. U.S.A. 85:208‐212.
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