E. coli Plasmid Vectors: Methods and ApplicationsNicola Casali, Andrew Preston A comprehensive collection of readily reproducible techniques for the manipulation of recombinant plasmids using the bacterial host E. coli. The authors describe proven methods for cloning DNA into plasmid vectors, transforming plasmids into E. coli, and analyzing recombinant clones. They also include protocols for the construction and screening of libraries, as well as specific techniques for specialized cloning vehicles, such as cosmids, bacterial artificial chromosomes, l vectors, and phagemids. Common downstream applications such as mutagenesis of plasmids, recombinant protein expression, and the use of reporter genes, are also described. |
Contents
HighPurity Plasmid Isolation Using Silica Oxide | 83 |
Isolation of Cosmid and BAC DNA from E coli | 99 |
Using Desktop Cloning Software to Plan Track | 107 |
Cloning in Plasmid Vectors | 121 |
Extraction of DNA from Agarose Gels | 137 |
Construction of Genomic Libraries in λVectors | 153 |
Rapid Screening of Recombinant Plasmids | 169 |
Restriction Analysis of Recombinant Plasmids | 175 |
SiteDirected Mutagenesis Using the Megaprimer Method | 203 |
Creating Nested DNA Deletions Using Exonuclease III | 225 |
Transposon and Transposome Mutagenesis | 233 |
In Vitro Transcription and Translation | 247 |
Vectors for the Expression of Recombinant Proteins in E coli | 257 |
Expression of Recombinant Proteins from lac Promoters | 277 |
Assays for βGalactosidase | 289 |
Assays for Green | 297 |
Other editions - View all
E. coli Plasmid Vectors: Methods and Applications Nicola Casali,Andrew Preston No preview available - 2010 |
E. Coli Plasmid Vectors: Methods and Applications Nicola Casali,Andrew Preston No preview available - 2003 |
Common terms and phrases
acetate agar agarose gel agarose gel electrophoresis aliquots antibiotic assay autoclave bacterial bacteriophage Biol cells Centrifuge Chapter chloroform chromosome coli Plasmid Vectors colonies conjugation containing copy number cosmid culture described in Subheading dilution DNA fragments DNA ligase DNA polymerase EDTA efficiency electroporation encoded Escherichia coli ethanol exonuclease expression extract fluorescence gene genetic genomic genomic DNA host strain hybridization Incubate at 37°C insert DNA IPTG isoamyl alcohol lac operon ligation membrane mg/mL Microbiol microcentrifuge Molecular Biology mutagenesis mutation Note nucleotides operon overhangs PCR product pellet phage phenol pipet plasmid DNA plate prepared primers probe Promega promoter Protocols purification reaction buffer recombinant replication restriction digestion restriction enzyme Resuspend room temperature sample screening solution sterile Store at 20°C supernatant T-vector T4 DNA target DNA template tion transcription transfer transformation transposon Tris-HCl pH tube vector DNA vitro volume