PCR Cloning Protocols

Front Cover
Bing-Yuan Chen, Harry W. Janes
Springer Science & Business Media, Feb 5, 2008 - Science - 439 pages
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.

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Contents

Computer Programs for PCR Primer Design and Analysis
19
SingleStep PCR Optimization
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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