PCR Cloning ProtocolsBing-Yuan Chen, Harry W. Janes PCR Cloning Protocols, Second Edition, updates and expands Bruce White's best-selling PCR Cloning Protocols (1997) with the newest procedures for DNA cloning and mutagenesis. Here the researcher will find readily reproducible methods for all the major aspects of PCR use, including PCR optimization, computer programs for PCR primer design and analysis, and novel variations for cloning genes of special characteristics or origin, with emphasis on long distance PCR and GC-rich template amplification. Also included are both conventional and novel enzyme-free and restriction site-free procedures to clone PCR products into a range of vectors, as well as state-of-the-art protocols to facilitate DNA mutagenesis and recombination, and to clone the challenging uncharacterized DNA flanking a known DNA fragment. |
From inside the book
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Page 10
... note that the annealing salt concentration temperature. affects the Tm of the primer:template duplex, and hence the 1.10.4. Cosolvents A variety of PCR cosolvents have been utilized to increase the yield, efficacy, and specificity of ...
... note that the annealing salt concentration temperature. affects the Tm of the primer:template duplex, and hence the 1.10.4. Cosolvents A variety of PCR cosolvents have been utilized to increase the yield, efficacy, and specificity of ...
Page 15
... Notes 1. Even though the PCR process has greatly enhanced scientific studies, a variety of prob- lems with the process ... Note, an increase in primer-dimer formation will decrease the production of the desired product. Third, Taq DNA ...
... Notes 1. Even though the PCR process has greatly enhanced scientific studies, a variety of prob- lems with the process ... Note, an increase in primer-dimer formation will decrease the production of the desired product. Third, Taq DNA ...
Page 21
... Note 2). Selected computer programs for nondegenerate PCR primer design and their features are listed in Table 1. From a computational point of view the design of nondegenerate PCR primers is relatively simple: find short substrings ...
... Note 2). Selected computer programs for nondegenerate PCR primer design and their features are listed in Table 1. From a computational point of view the design of nondegenerate PCR primers is relatively simple: find short substrings ...
Page 26
... Note 6 ) . 2. Replace the default URL address with http://bibiserv.techfak.unibielefeld.de/genefisher/ and hit return . After connection , GeneFisher Interactive PCR Primer Design home page will appear in your browser . 3. Click Start ...
... Note 6 ) . 2. Replace the default URL address with http://bibiserv.techfak.unibielefeld.de/genefisher/ and hit return . After connection , GeneFisher Interactive PCR Primer Design home page will appear in your browser . 3. Click Start ...
Page 32
... Note 2 ) . Mismatching might occur when attempting to amplify specific members from a complex multigene family , when using nucleotide sequence information deduced from an amino acid sequence , or when amplifying across species lines ...
... Note 2 ) . Mismatching might occur when attempting to amplify specific members from a complex multigene family , when using nucleotide sequence information deduced from an amino acid sequence , or when amplifying across species lines ...
Contents
19 | |
31 | |
Lori A Kolmodin | 37 |
Coupled OneStep Reverse Transcription and Polymerase Chain | 53 |
Long Distance ReverseTranscription | 59 |
from ParaffinEmbedded Tissues | 67 |
Teruaki Tozaki 10 MethylationSpecific PCR | 81 |
Haruhiko Ohashi | 91 |
A Fast Polymerase Chain ReactionMediated Strategy for Introducing | 217 |
PCR Screening in SignatureTagged Mutagenesis of Essential Genes | 225 |
Staggered Extension Process StEP In Vitro Recombination | 235 |
PCRBased Strategies to Clone Unknown DNA Regions | 249 |
Long Distance Vectorette PCR LDV PCR | 275 |
Nonspecific Nested Suppression PCR Method | 285 |
cDNA Cloning | 293 |
Genomic DNA Cloning | 301 |
Contents | 99 |
Contents ix | 103 |
CLONING PCR PRODUCTS | 111 |
Using T4 DNA Polymerase to Generate Clonable PCR Products | 121 |
Directional Restriction SiteFree Insertion of PCR Products | 133 |
A Rapid and Simple Procedure for Direct Cloning | 153 |
MUTAGENESIS AND RECOMBINATION | 167 |
InFrame Cloning of Synthetic Genes Using PCR Inserts | 175 |
Megaprimer | 189 |
PCR Method for Generating Multiple Mutations at Adjacent Sites | 207 |
Gene Cloning and Expression Profiling by Rapid Amplification | 309 |
The Isolation of DNA Sequences Flanking Tn5 Transposon Insertions | 315 |
Rapid Amplification of Genomic DNA Sequences Tagged | 325 |
by Anchored | 337 |
A Step Down PCRBased Technique for Walking | 343 |
Cloning of Homologous Genes by GeneCapture | 359 |
Rapid and Nonradioactive Screening of Recombinant Libraries by | 377 |
Generation and PCR Screening of Bacteriophage λ Sublibraries | 391 |
A 384Well MicrotiterPlateBased Template Preparation | 411 |
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Common terms and phrases
Acids Res agarose gel electrophoresis aliquot amplicons analysis annealing temperature approx AS-PCR baculovirus BioTechniques blunt-end cDNA cells centrifuge cloning method cloning of PCR coli concentration containing denaturation digestion direct cloning DNA fragments DNA ligase DNA sequence dNTP double-stranded EDTA efficiency ethanol ethidium bromide flanking full-length genomic DNA H. W. Janes hybridization in-frame cloning Incubate insert inverse PCR IPCR isolation known sequence ligation reaction megaprimer Meth MgCl2 mixture mutagenesis mutations Note Nucl nucleotide oligonucleotide PCR amplification PCR buffer PCR Cloning Protocols PCR primers PCR products plasmid plasmid DNA plate polymerase chain reaction primer primer design probe protein purified Qiagen reagents recombination region restriction endonuclease restriction enzyme restriction sites room temperature sample screening single-stranded specific step sterile strand strategy Subheading synthesis Taq DNA polymerase Taq polymerase target technique template thermal cycler tion transposon Tris-HCl tube virus vitro