DNA Cloning: A Hands-on Approach

Seok-Yong Choi, Hyunju Ro, Hankuil Yi (Autoren)

Buch | Hardcover

131 Seiten

2019 | 1st ed. 2019
Springer (Verlag)
978-94-024-1660-2 (ISBN)

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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.

Seok-Yong Choi, Department of Biomedical Sciences, Chonnam National University College of Medicine, Gwangju, Republic of Korea Hyunju Ro, Department of Biological Sciences, Chungnam National University, Daejeon, Republic of Korea Hankuil Yi, Department of Biological Sciences, Chungnam National University, Daejeon, Republic of Korea

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-directedmutagenesis.- 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.

Erscheinungsdatum	02.05.2019
Zusatzinfo	70 Illustrations, color; 23 Illustrations, black and white; XIII, 131 p. 93 illus., 70 illus. in color.
Verlagsort	Dordrecht
Sprache	englisch
Maße	155 x 235 mm
Themenwelt	Medizin / Pharmazie ► Medizinische Fachgebiete
	Studium ► 2. Studienabschnitt (Klinik) ► Humangenetik
	Naturwissenschaften ► Biologie ► Botanik
	Naturwissenschaften ► Biologie ► Genetik / Molekularbiologie
	Technik ► Umwelttechnik / Biotechnologie
Schlagworte	Cloning • DNA • PCR • Plasmid • Vector
ISBN-10	94-024-1660-9 / 9402416609
ISBN-13	978-94-024-1660-2 / 9789402416602
Zustand	Neuware