UID:
almafu_9959328692002883
Format:
1 online resource (xiii, 214 pages) :
,
illustrations
Edition:
Electronic reproduction. [Place of publication not identified] : HathiTrust Digital Library, 2010.
ISBN:
3527603697
,
9783527603695
,
3527604626
,
9783527604623
,
1280519916
,
9781280519918
Content:
Miniturization and high throughput assay technology have brought the power of molecular evolution to the bioscience laboratory. Applied wisely, the evolutionary approach can quickly yield the desired result even where other methods have failed. From library generation by random or directed mutagenesis to screening and selection techniques--the crucial steps for successful evolutionary biotechnology are described in detail in this practical guide that also includes valuable troubleshooting hints on frequently encountered problems. Modern methods for the surface display of peptides and proteins.
Note:
Cover -- Contents -- List of Contributors -- 1 Introduction -- References -- 2 Generation of Mutant Libraries Using Random Mutagenesis -- 2.1 Introduction -- 2.2 Materials -- 2.2.1 Materials for Random PCR Mutagenesis -- 2.2.2 Materials for Mutator Strain Passage -- 2.3 Protocols -- 2.3.1 Protocol for Random PCR Mutagenesis According to Joyce -- 2.3.2 Protocol for Mutator Strain Passage -- 2.4 Troubleshooting -- References -- 3 DNA Shuffling -- 3.1 Introduction -- 3.2 Materials -- 3.2.1 For Preparation of Parental Genes -- 3.2.2 For Random Fragmentation by DNase I -- 3.2.3 For Collection of DNA Fragments in Specific Molecular Size Ranges -- 3.2.4 For Reassembly of These Fragments by Primerless PCR -- 3.2.5 For Amplification of Reassembled Products by Conventional PCR with Primers -- 3.3 Protocol -- 3.3.1 Preparation of Parental Genes -- 3.3.2 Random Fragmentation by DNase I -- 3.3.3 Collection of DNA Fragments in Specific Molecular Size Ranges -- 3.3.4 Reassembly of These Fragments by Primerless PCR -- 3.3.5 Amplification of Reassembled Products by Conventional PCR with Primers -- 3.4 Troubleshooting -- 3.4.1 Insufficient DNase I Fragmentation -- 3.4.2 Little or No Product of Primerless PCR -- 3.4.3 Little or No Product of PCR with Primers -- 3.4.4 The Product of PCR with Primers is Multi-banded -- 3.5 Amplification Examples -- References -- 4 DNA Recombination Using StEP -- 4.1 Introduction -- 4.2 Materials -- 4.2.1 StEP PCR -- 4.2.2 Purification of an Appropriate DNA Fragment -- 4.2.3 Equipment -- 4.3 Protocol -- 4.4 Technical Tips -- 4.4.1 Problem: Little or No PCR Product (Full-length Product) after PCR -- 4.4.2 Problem: High Background Levels of DNA after PCR -- 4.5 StEP in Directed Evolution -- References -- 5 FACS Screening of Combinatorial Peptide and Protein Libraries Displayed on the Surface of Escherichia coli Cells -- 5.1 Introduction -- 5.2 Materials -- 5.2.1 Escherichia coli Strains and Plasmids -- 5.2.2 Liquid Media and Agar Plates -- 5.2.3 Biological and Chemical Materials -- 5.2.4 Equipment -- 5.3 Protocols -- 5.3.1 Verification of Cell Surface Exposure of the Passenger Protein -- 5.3.2 Labeling of the Target Protein -- 5.3.3 Library Construction -- 5.3.4 Combinatorial Library Screening by FACS and MACS -- 5.4 Troubleshooting -- 5.5 Major Applications -- References -- 6 Selection of Phage-displayed Enzymes -- 6.1 Introduction -- 6.2 Materials -- 6.2.1 Buffers, Reagents and Consumables -- 6.2.2 Strains and Vectors -- 6.3 Protocols -- 6.3.1 The Phage-enzyme -- 6.3.2 Library Construction -- 6.3.3 Selection -- 6.4 Troubleshooting -- 6.4.1 Phage Titers Are Not Reproducible -- 6.4.2 Phage-enzymes Degrade with Time -- 6.4.3 Phages Are Not Genetically Stable.
,
Master and use copy. Digital master created according to Benchmark for Faithful Digital Reproductions of Monographs and Serials, Version 1. Digital Library Federation, December 2002.
Additional Edition:
Print version: Brakmann, Susanne. Evolutionary methods in biotechnology. Weinheim : Wiley-VCH, ©2004 ISBN 3527307990
Additional Edition:
ISBN 9783527307999
Language:
English
Keywords:
Electronic books.
;
Electronic books.
;
Electronic books.
URL:
https://onlinelibrary.wiley.com/doi/book/10.1002/3527603697
URL:
https://onlinelibrary.wiley.com/doi/book/10.1002/3527603697
URL:
https://onlinelibrary.wiley.com/doi/book/10.1002/3527603697
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