Cloning genomic sequences using long-range PCR |
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Authors: | John Mundy Raphael Mayer Nam-Hai Chua |
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Affiliation: | (1) Laboratory of Plant Molecular Biology, Rockefeller University, 1230 York Avenue, 10021 New York, N.Y., USA;(2) Department of Plant Physiology, Copenhagen University, Oster Farimagsgade 2A, 1353 Copenhagen K, Denmark |
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Abstract: | Protocols are presented for preparing DNA from a genomic library in λ phage and for synthesizing genomic fragments using PCR with nested vector- and gene-specific primers and linker-primers. Library DNA, isolated fromE. coli liquid lysates by a simple protocol, is used as template in PCR following a commercial protocol. The method produces library DNA sufficient for several hundred PCRs, incorporates nested primers to reduce nonspecific product formation, and enables the synthesis of linker-containing DNA fragments containing selected restriction sites to simplify subsequent cloning. The isolation of 5′ upstream sequences of three different arabidopsis genes by this methodod is described. |
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Keywords: | Arabidopsis thaliana gene gibberellic acid inverse PCR lambda phage promoter |
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