Methods in Enzymology Vol.154 Recombinant DNA, Part E

This chapter describes detailed procedures for the construction of full-length complementary deoxyribo nucleic acid (cDNA) expression libraries and the screening of the libraries for particular clones based on their transient expression in mammalian cells. Methods for library transduction and screening based on stable expression are described in this chapter. In the steps that have just been enumerated, double-stranded, full length deoxyribo nucleic acid (DNA) copies of the original messenger ribo nucleic acids (mRNAs) are efficiently synthesized and inserted into the vector to form a functional composite gene with the protein coding sequence derived from the cDNA and the transcriptional and RNA processing signals from the SV40 genome. Successful construction of full-length cDNA libraries depends heavily on the quality of the mRNA preparation. The use of intact, uncontaminated mRNA is essential for generating full-length clones.To screen for or selects a particular clone on the basis of the function it encodes, the library is acutely transfected or stably transduced into cultured cells. The screening method relies on transient expression of cDNA clones in mammalian cells.

• Series: Methods in Enzymology (Book 154)
• Hardcover: 576 pages
• Publisher: Academic Press;
• Language: English
• ISBN-10: 0121820556
• ISBN-13: 978-0121820558
• Product Dimensions: 6 x 1.3 x 9 inches

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