Production of a Complete cDNA Library

Lloyd B. Klickstein1

1 Brigham and Women's Hospital, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 5.8A
DOI:  10.1002/0471142727.mb0508as18
Online Posting Date:  May, 2001
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Abstract

A complete cDNA library is one that contains at least one cDNA clone representing each mRNA in the cell. A method is provided in this unit for calculating the required base of a cDNA library such that the library should contain at least one copy of every mRNA. Either a phage or plasmid vector can be used in constructing a complete cDNA library, and protocols are described for both. Procedures for evaluating these cDNA libraries are also provided along with a procedure for evaluating the quality of the λ arms used in constructing complete phage cDNA libraries.

     
 
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Table of Contents

  • Basic Protocol 1: Ligation and Transfection for a Complete λ Phage Library
  • Alternate Protocol 1: Ligation and Transformation for a Complete Plasmid Library
  • Support Protocol 1: Evaluation of a cDNA Library
  • Support Protocol 2: Preparation of Test Insert for Evaluation of Phosphatased Phage Arms
  • Commentary
  • Tables
     
 
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Materials

Basic Protocol 1: Ligation and Transfection for a Complete λ Phage Library

  Materials
  • EcoRI‐cut, phosphatased λgt10, λgt11, or λgt11 DNA or other phage vector DNA
  • Double‐stranded cDNA with EcoRI ends (unit 5.6)
  • 10× DNA ligase buffer (unit 3.4)
  • T4 DNA ligase (measured in cohesive‐end units; unit 3.14)
  • Cold 10 mM MgSO 4
  • λ phage packaging extract ( Stratagene)
  • Suspension medium (SM; unit 1.11)
  • Chloroform
  • E. coli C600hflA(λgt10) or Y1088(λgt11)
  • IPTG (λgt11 only; unit 1.4)
  • Xgal (λgt11 only; unit 1.4)
  • Additional reagents and equipment for plating and titering bacteriophage (unit 1.11)

Alternate Protocol 1: Ligation and Transformation for a Complete Plasmid Library

  Additional Materials
  • CDM8 plasmid DNA ( Invitrogen) or similar plasmid vector DNA, CsCl‐purified
  • BstXI and 10× BstXI buffer (unit 3.1)
  • Low melting temperature agarose for fragment purification (unit 2.6)
  • TE buffer ( appendix 22)
  • Double‐stranded cDNA with BstXI ends (unit 5.6)
  • Competent cells: MC1061/P3 (Invitrogen) is required for CDM8; MC1061, DK1, DH1, DH5, or HB101 are suitable for other vectors
  • LB medium (unit 1.1)
  • 150‐mm LB agar plates (unit 1.1)with appropriate antibiotics (10 µg/ml ampicillin + 7.5 µg/ml tetracycline for CDM8 in MC1061/P3; 50 to 100 µg/ml ampicillin for the other vectors and strains above)
  • 80% glycerol in H 2O
  • Additional reagents and equipment for plating and growth of E. coli (units 1.1 1.4), isolation of DNA from agarose gels (unit 2.6), alkaline lysis/CsCl gradient preparation of plasmid DNA (unit 1.7), and introduction of plasmid DNA into cells (unit 1.8)

Support Protocol 1: Evaluation of a cDNA Library

  Additional Materials
  • Genomic DNA
  • 1:1 phenol/chloroform (unit 2.1)
  • Additional reagents and equipment for restriction endonuclease digestions (unit 3.1) and plating bacteriophage libraries (unit 6.1)
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Figures

Videos

Literature Cited

Literature Cited
   Huynh, T.V., Young, R.A., and Davis, R.W. 1984. Constructing and screening cDNA libraries in λgt10 and λgt11. In DNA Cloning Techniques, Vol. 1: A Practical Approach (D. Glover, ed.) pp. 49‐78. IRL Press, Oxford.
   Okayama, H. and Berg, P. 1982. High‐efficiency cloning of full‐length cDNA. Mol. Cell. Biol. 2:161‐170.
   Sambrook, J., Maniatis, T., and Fritsch, E.F. 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
   Seed, B. 1983. Purification of genomic sequence from bacteriophage libraries by recombination and selection in vivo. Nucl. Acids Res. 12:5627‐5633.
   Seed, B. 1987. An LFA‐ 3 cDNA encodes a phospholipid‐linked membrane protein homologous to its receptor CD2. Nature (Lond.) 329:840‐842.
   Young, R.A. and Davis, R.W. 1983. Efficient isolation of genes by using antibody probes. Proc. Natl. Acad. Sci. U.S.A. 80:1194‐1198.
Key References
   Huynh et al., 1984. See above.
  Describes production of a complete λ phage library.
   Seed, B., 1987. See above.
  Describes production and screening of a complete plasmid library.
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