SpringerProtocols: Abstract: cDNA Library Construction for the Lambda ZAP®-Based Vectors

cDNA Library Construction for the Lambda ZAP®-Based Vectors

By: Marjory A. Snead², Michelle A. Alting-Mees³, Jay M. Short²

Abstract

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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.

Affiliation(s): (2) Recombinant BioCatalysis Inc., La Jolla, CA
(3) Stratagene Cloning Systems, La Jolla, CA

Book Title: cDNA Library Protocols

Series: Methods in Molecular Biology | Volume: 69 | Pub. Date: Oct-01-1996 | Page Range: 39-51 | DOI: 10.1385/0-89603-383-X:39

Subject: Genetics/Genomics

References

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