BookSeok-Yong Choi, Hyunju Ro, Hankuil Yi.
Summary: This book offers step-by-step instruction on DNA cloning, defined as moving genes around plasmids, mutating genes, or mining new genes. The aim is to provide those new to the field with reliable and up-to-date practical guidance while at the same time conveying the scope for creativity. After a brief synopsis of the history of cloning, the fundamentals and prerequisites are explained, covering, for example, software, vectors commonly used in the lab, appropriate choice of restriction endonucleases, the preparation of agarose gels, competent cells, and LB agar plates, and procedures to be followed upon receipt of new plasmids. The remainder of the book is devoted to the clear description of methods and individual steps in cloning. Guidance is provided on the cut and paste method, DNA sequencing, direct sequencing, primer design, PCR-based gene insertion and deletion, epitope tag insertion, the use of RACE technology, BAC recombineering, and much, much more. Sources of error and a variety of techniques that make life considerably easier when cloning are also examined in detail.
Contents:
Part I. What is cloning?
Chapter 1.1 Definition of cloning
Chapter 1.2 Discovering a new gene
Chapter 1.3 Cloning in the past
Chapter 1.4 Cloning in the present
Part II. A prerequisite for cloning
Chapter 2.1 Software useful for cloning design
Chapter 2.2 Vector, plasmid, construct and the Kozak consensus sequence
Chapter 2.3 Multiple cloning sites (MCS)
Chapter 2.4 Restriction endonucleases and star activity
Chapter 2.5 Agarose gel electrophoresis
Chapter 2.6 Pouring LB agar plates
Chapter 2.7 Competent cells
Chapter 2.8 The conversion of DNA mass into molar concentration
Chapter 2.9 Upon receiving new plasmids
Chapter 2.10 cDNA library
Part III. The first step in cloning
Chapter 3.1 Cut & paste
Chapter 3.2 DNA sequencing and direct sequencing
Chapter 3.3 PCR and nested PCR
Chapter 3.4 Fill-in (Full & Partial)
Chapter 3.5 Compatible cohesive ends
Chapter 3.6 Methylation
Chapter 3.7 Three-piece ligation
Chapter 3.8 Site-directed mutagenesis
Chapter 3.9 Structure of plant transformation vectors
Chapter 3.10 Transformation of R. radiobacter
Part IV. The next step of cloning
Chapter 4.1 How to insert a DNA fragment into a gene
Chapter 4.2 How to delete an internal region of a gene
Chapter 4.3 How to insert an epitope tag into a gene
Chapter 4.4 Translational fusion vs. transcriptional fusion
Part V. The last steps of cloning
Chapter 5.1 Method for cloning similar genes in different species
Chapter 5.2 RACE (rapid amplification of cDNA ends)
Chapter 5.3 BAC recombineering
Chapter 5.4 Old trick: partial digestion
Chapter 5.5 Modification of a vector
Chapter 5.6 When you notice a frame shift mutation upon cloning
Chapter 5.7 Reality of cloning: an extremely unlucky case
Part VI. Methods that make your cloning life easier
Chapter 6.1 TA cloning and production of a T-vector
Chapter 6.2 TOPO TA cloning
Chapter 6.3 Gateway cloning
Chapter 6.4 Golden Gate Assembly for a modular cloning
Chapter 6.5 In-Fusion Sequence and Ligation-Independent Cloning (In-Fusion SLIC)
Chapter 6.6 T4 DNA Polymerase Sequence-and Ligation-Independent Cloning (T4 DNA Pol SLIC)
Chapter 6.7 Non-template PCR cloning
Part VII. Advice to cloners
Chapter 7.1 When cloning is not going well
Chapter 7.2 Keep your cloning data organized
Appendix 1. Further readings
Appendix 2. Abbreviations
Appendix 3. Index.