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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Abbreviations
- LB:
-
Luria-Bertani medium
- PCR:
-
polymerase chain reaction
- PEG:
-
polyethylene glycol
- TAE:
-
tris-acetate buffer
- UTR:
-
untranslated sequence
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Mundy, J., Mayer, R. & Chua, NH. Cloning genomic sequences using long-range PCR. Plant Mol Biol Rep 13, 156–163 (1995). https://doi.org/10.1007/BF02668787
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DOI: https://doi.org/10.1007/BF02668787