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cDNA Library Construction for the Lambda ZAP®-Based Vectors

  • Marjory A. Snead
  • Michelle A. Alting-Mees
  • Jay M. Short
Protocol
  • 94 Downloads
Part of the Springer Protocols Handbooks book series (SPH)

Abstract

Each organism and tissue type has a unique population of messenger RNA (mRNA) molecules. These mRNA populations are difficult to maintain, clone, and amplify; therefore, they must be converted to more stable DNA molecules (cDNA). Successful cDNA synthesis should yield full-length copies of the original population of mRNA molecules. Hence, the quality of the cDNA library can be only as good as the quality of the mRNA. Pure, undegraded mRNA is essential for the construction of large, representative cDNA libraries (1). Secondary structure of mRNA molecules can cause the synthesis of truncated cDNA fragments. In this case, treatment of the mRNA with a denaturant, such as methyl-mercuric hydroxide, prior to synthesis may be necessary (2). Other potential difficulties include DNA molecules contaminating the mRNA sample. DNA can clone efficiently, and their introns can confuse results. RNase-free DNase treatment of the sample is recommended.

Keywords

mRNA Molecule mRNA Sample Geiger Counter Unincorporated Nucleotide Ligation Buffer 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Humana Press Inc., Totowa, NJ 2000

Authors and Affiliations

  • Marjory A. Snead
    • 1
  • Michelle A. Alting-Mees
    • 2
  • Jay M. Short
    • 1
  1. 1.Diversa CorporationSan Diego
  2. 2.Stratagene CorporationLa Jolla

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