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
... thermal transfer characteristics. Typically, polypropylene is used for PCR vessels and conventional, thick-walled microcentrifuge tubes are chosen for many thermal cycler systems. PCR is most often performed at a 10–100 μL reaction ...
... thermal transfer characteristics. Typically, polypropylene is used for PCR vessels and conventional, thick-walled microcentrifuge tubes are chosen for many thermal cycler systems. PCR is most often performed at a 10–100 μL reaction ...
Page 12
... thermal cyclers have been designed that use a heated cover over the tubes ... thermal cycle. If insufficient hold time is specified at any temperature, the ... cycler controlled by block tempera- ture, a 60-s hold time is sufficient for ...
... thermal cyclers have been designed that use a heated cover over the tubes ... thermal cycle. If insufficient hold time is specified at any temperature, the ... cycler controlled by block tempera- ture, a 60-s hold time is sufficient for ...
Page 13
... Thermal Cycler (PE Applied Biosystems GeneAmp PCR System). 3. Methods 3.1. Hot Start Process In the AmpliTaq Gold Hot Start process (33), a master mix is prepared at room temperature, aliquoted into individual tubes, and thermal cycled ...
... Thermal Cycler (PE Applied Biosystems GeneAmp PCR System). 3. Methods 3.1. Hot Start Process In the AmpliTaq Gold Hot Start process (33), a master mix is prepared at room temperature, aliquoted into individual tubes, and thermal cycled ...
Page 14
... PCR amplifications within a programmed thermal cycler . For the Perkin Elmer DNA Thermal Cycler 9600 , program and run the following linked files : a . CYCL File : 95 ° C for 9 min , 1 cycle ; link to file ( b ) . b . CYCL File : 94 ° C ...
... PCR amplifications within a programmed thermal cycler . For the Perkin Elmer DNA Thermal Cycler 9600 , program and run the following linked files : a . CYCL File : 95 ° C for 9 min , 1 cycle ; link to file ( b ) . b . CYCL File : 94 ° C ...
Page 22
... PCR Wizard” allows you to design primers to any given DNA template sequence as well as to generate a Techne Genius Thermal Cycler pro- gram file, which can be sent from a PC directly to multiple Genius thermal cyclers (up to 32) ...
... PCR Wizard” allows you to design primers to any given DNA template sequence as well as to generate a Techne Genius Thermal Cycler pro- gram file, which can be sent from a PC directly to multiple Genius thermal cyclers (up to 32) ...
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