cDNA Library Protocols

1. Front Matter

   Pages i-x

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2. cDNA Library Construction from Small Amounts of RNA Using Paramagnetic Beads and PCR

   • Kris N. Lambert, Valerie M. Williamson

   Pages 1-12

3. Increasing the Average Abundance of Low-Abundance cDNAs by Ordered Subdivision of cDNA Populations

   • David R. Sibson, Michael P. Starkey

   Pages 13-32

4. Isolation of Messenger RNA from Plant Tissues

   • Alison Dunn

   Pages 33-38

5. cDNA Library Construction for the Lambda ZAP®-Based Vectors

   • Marjory A. Snead, Michelle A. Alting-Mees, Jay M. Short

   Pages 39-51

6. Clone Excision Methods for the Lambda ZAP®-Based Vectors

   • Marjory A. Snead, Michelle A. Alting-Mees, Jay M. Short

   Pages 53-60

7. Using Rapid Amplification of cDNA Ends (RACE) to Obtain Full-Length cDNAs

   • Yue Zhang, Michael A. Frohman

   Pages 61-87

8. Inverse PCR Approach to Cloning cDNA Ends

   • Sheng-He Huang

   Pages 89-96

9. Cloning Gene Family Members Using the Polymerase Chain Reaction with Degenerate Oligonucleotide Primers

   • Gregory M. Preston

   Pages 97-113

10. Subtractive Hybridization for the Isolation of Differentially Expressed Genes Using Magnetic Beads

    • Hans-Christian Aasheim, Ton Logtenberg, Frank Larsen

    Pages 115-128

11. Preparation of Competent Cells for High-Efficiency Plasmid Transformation of Escherichia coli

    • Yasuhiko Nakata, Xiaoren Tang, Kazunari K. Yokoyama

    Pages 129-137

12. The Tetramethylammonium Chloride (TMAC) Method for Screening cDNA Libraries with Highly Degenerate Oligonucleotide Probes Obtained by Reverse Translation of Amino Acid Sequences

    • Bent Honoé, Peder Madsen

    Pages 139-146

13. Screening cDNA Libraries by Hybridization with Double-Stranded DNA Probes and Oligonucleotides

    • Caroline A. Austin

    Pages 147-154

14. Immunological Screening of λ-Phage cDNA Expression Libraries

    • Helen C. Hurst

    Pages 155-159

15. Cloning Sequence-Specific DNA-Binding Factors from cDNA Expression Libraries Using Oligonucleotide Binding Site Probes

    • Ian G. Cowell

    Pages 161-170

16. Protein Interaction Cloning by Far-Western Screening of λ-Phage cDNA Expression Libraries

    • Shinichi Takayama, John C. Reed

    Pages 171-184

17. Yeast Two-Hybrid Library Screening

    • Ian G. Cowell

    Pages 185-202

18. Signal Sequence Trap

    • Kei Tashiro, Toru Nakano, Tasuku Honjo

    Pages 203-219

19. Isolation of Genetic Suppressor Elements (GSEs) from Random Fragment cDNA Libraries in Retroviral Vectors

    • Andrei V. Gudkov, Igor B. Roninson

    Pages 221-240

20. Expression and Preparation of Fusion Proteins from Recombinant λgt1.1 Phages

    • Sheng-He Huang, Ambrose Jong

    Pages 241-245

The first libraries of complementary DNA (cDNA) clones were con­ structed in the mid-to-late 1970s using RNA-dependent DNA polymerase (reverse transcriptase) to convert poly A* mRNA into double-stranded cDNA suitable for insertion into prokaryotic vectors. Since then cDNA technology has become a fundamental tool for the molecular biologist and at the same time some very significant advances have occurred in the methods for con­ structing and screening cDNA libraries. It is not the aim of cDNA Library Protocols to give a comprehensive review of all cDNA library-based methodologies; instead we present a series of up-to-date protocols that together should give a good grounding of proce­ dures associated with the construction and use of cDNA libraries. In deciding what to include, we endeavored to combine up-to-date versions of some of the most widely used protocols with some very usefiil newer techniques. cDNA Library Protocols should therefore be especially useful to the investigator who is new to the use of cDNA libraries, but should also be of value to the more experienced worker. Chapters 1—5 concentrate on cDNA library construction and manipula­ tion, Chapters 6 and 7 describe means of cloning difficult-to-obtain ends of cDNAs, Chapters 8-18 give various approaches to the screening of cDNA libraries, and the remaining chapters present methods of analysis of cDNA clones including details of how to analyze cDNA sequence data and how to make use of the wealth of cDNA data emerging from the human genome project.

This is a good overview of the essentials for preparing a cDNA library, for both the novice and the expert...the book is a true laboratory manual for the experimental scientist. It Should represent a useful and up-to-date addition for any lab that, for whatever reason, needs to generate its own libraries.-Hoffman-LaRoche, Inc.

"...their fine layout and ease of use suggest that the lab copy won't stay on the bookshelf for long."-SGM Quarterly

• University of Newcastle, UK

  Ian G. Cowell, Caroline A. Austin

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