By Lori A. Kolmodin, David E. Birch (auth.), Bing-Yuan Chen, Harry W. Janes (eds.)
In the post-genomic period, PCR has turn into the tactic of selection not just for cloning latest genes, but in addition for producing a wide range of novel genes by means of mutagenesis and/or recombination in the genes of curiosity. PCR Cloning Protocols, moment version, updates and expands Bruce White's best-selling PCR Cloning Protocols (1997) with the most recent tactics for DNA cloning and mutagenesis. the following the researcher will locate without difficulty reproducible tools for the entire significant facets of PCR use, together with PCR optimization, machine courses for PCR primer layout and research, and novel diversifications for cloning genes of precise features or beginning, with emphasis on long-distance PCR and GC-rich template amplification. additionally integrated are either traditional and novel enzyme-free and limit site-free methods to clone PCR items right into a diversity of vectors, in addition to state of the art protocols to facilitate DNA mutagenesis and recombination and to clone the tough uncharacterized DNA flanking a recognized DNA fragment. robust purposes of PCR in library building and sublibrary new release and screening also are provided.
Authoritative and up to date, PCR Cloning Protocols, moment variation, constitutes a gold-standard number of the quickest, easiest, and most well-liked tools for keeping apart genes from all organic samples and growing novel genes from them via mutagenesis/recombination-essential tools for brand new examine of useful genomics, gene expression, protein structure-function relationships, protein engineering, and molecular evolution.
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Additional resources for PCR Cloning Protocols
Jhu. techfak. de/genefisher/ CODEHOP Internet Browser Free Design degenerate PCR primers from protein multiple sequence alignments. blocks. asp NetPrimer Internet Browser Free Java Applet Analyze basic properties and second structures for an individual primer or primer pair. premierbiosoft. html 3. 1. Designing Nondegenerate PCR Primers Using Primer3 Primer3 was developed at Whitehead Institute for Biomedical Research and Howard Hughes Medical Institute. It contains so many parameters that most people only need a subset of them to use as the criteria for primer selection.
7. 5% agarose gel electrophoresis. 2. Stepdown PCR with a Mismatched Degenerate Primer 1. Template DNA (rabbit genomic liver 125 ng/µL). 2. Primers: Prepare stock solutions at 200 µg/mL. The following primer pair yields a 703-bp amplicon. The sites of mismatches are in capital letters. Degeneracies are separated by a slash and are in parenthesis. The sequence of the genomic homologous strand corresponding to the primer with degeneracies is presented (shown in brackets) for comparative purposes.
H. (1995) The use of cosolvents to enhance amplification by the polymerase chain reaction, in PCR Strategies (Innes, M. , Gelfand, D. , and Sninsky, J. ), Academic, San Diego, CA, pp. 3–16. 27. , Jung, K. , and Loening, S. (1997) Betaine improves the PCR amplification of GC-rich DNA sequences. Nucl. Acids Res. 25 (19), 3957–3958. 28. , Gifford, J. , and Wilson, A. C. (1988) Mitochondrial DNA sequences from a 7000–year old brain. Nucl. Acids Res. 16, 9775–9787. 29. , and Sommer, S. S. (1990) Formamide can drastically increase the specificity of PCR.