From Genes to Genomes: Concepts and Applications of DNA Technology, Jeremy W. Dale, Malcolm von Schantz and Nick Plant. © 2012 John Wiley & Sons, Ltd. Chapter 2 How to Clone a Gene © 2012 John Wiley & Sons, Ltd.

From Genes to Genomes: Concepts and Applications of DNA Technology, Jeremy W. Dale, Malcolm von Schantz and Nick Plant. © 2012 John Wiley & Sons, Ltd. Figure 2.1 (a) Bacterial cloning and (b) gene cloning.

From Genes to Genomes: Concepts and Applications of DNA Technology, Jeremy W. Dale, Malcolm von Schantz and Nick Plant. © 2012 John Wiley & Sons, Ltd. Figure 2.2 Basic outline of gene cloning.

From Genes to Genomes: Concepts and Applications of DNA Technology, Jeremy W. Dale, Malcolm von Schantz and Nick Plant. © 2012 John Wiley & Sons, Ltd. Figure 2.3 Cutting and joining DNA.

From Genes to Genomes: Concepts and Applications of DNA Technology, Jeremy W. Dale, Malcolm von Schantz and Nick Plant. © 2012 John Wiley & Sons, Ltd. Figure 2.4 Ethanol precipitation. The tubes are spun in an angle rotor, so the precipitate is found at the side of the lower part of the tube.

From Genes to Genomes: Concepts and Applications of DNA Technology, Jeremy W. Dale, Malcolm von Schantz and Nick Plant. © 2012 John Wiley & Sons, Ltd. Figure 2.5 Alkaline denaturation procedure for plasmid purification.

From Genes to Genomes: Concepts and Applications of DNA Technology, Jeremy W. Dale, Malcolm von Schantz and Nick Plant. © 2012 John Wiley & Sons, Ltd. Figure 2.6 Analytical gel electrophoresis.

From Genes to Genomes: Concepts and Applications of DNA Technology, Jeremy W. Dale, Malcolm von Schantz and Nick Plant. © 2012 John Wiley & Sons, Ltd. Figure 2.7 Electrophoretic mobility of forms of plasmid DNA.

From Genes to Genomes: Concepts and Applications of DNA Technology, Jeremy W. Dale, Malcolm von Schantz and Nick Plant. © 2012 John Wiley & Sons, Ltd. Figure 2.8 Bacteriophage restriction.

From Genes to Genomes: Concepts and Applications of DNA Technology, Jeremy W. Dale, Malcolm von Schantz and Nick Plant. © 2012 John Wiley & Sons, Ltd. Figure 2.9 Reading a palindromic sequence.

From Genes to Genomes: Concepts and Applications of DNA Technology, Jeremy W. Dale, Malcolm von Schantz and Nick Plant. © 2012 John Wiley & Sons, Ltd. Figure 2.10 Sticky ends generated by EcoRI.

From Genes to Genomes: Concepts and Applications of DNA Technology, Jeremy W. Dale, Malcolm von Schantz and Nick Plant. © 2012 John Wiley & Sons, Ltd. Figure 2.11 Restriction fragment ends.

From Genes to Genomes: Concepts and Applications of DNA Technology, Jeremy W. Dale, Malcolm von Schantz and Nick Plant. © 2012 John Wiley & Sons, Ltd. Figure 2.12 Isoschizomers.

From Genes to Genomes: Concepts and Applications of DNA Technology, Jeremy W. Dale, Malcolm von Schantz and Nick Plant. © 2012 John Wiley & Sons, Ltd. Figure 2.13 Action of T4 DNA ligase.

From Genes to Genomes: Concepts and Applications of DNA Technology, Jeremy W. Dale, Malcolm von Schantz and Nick Plant. © 2012 John Wiley & Sons, Ltd. Figure 2.14 Ligation: some of the potential products.

From Genes to Genomes: Concepts and Applications of DNA Technology, Jeremy W. Dale, Malcolm von Schantz and Nick Plant. © 2012 John Wiley & Sons, Ltd. Figure 2.15 Ligation: effect of dephosphorylation of vector.

From Genes to Genomes: Concepts and Applications of DNA Technology, Jeremy W. Dale, Malcolm von Schantz and Nick Plant. © 2012 John Wiley & Sons, Ltd. Figure 2.16 Ligation using topoisomerase.

From Genes to Genomes: Concepts and Applications of DNA Technology, Jeremy W. Dale, Malcolm von Schantz and Nick Plant. © 2012 John Wiley & Sons, Ltd. Figure 2.17 Linkers.

From Genes to Genomes: Concepts and Applications of DNA Technology, Jeremy W. Dale, Malcolm von Schantz and Nick Plant. © 2012 John Wiley & Sons, Ltd. Figure 2.18 Adaptors.

From Genes to Genomes: Concepts and Applications of DNA Technology, Jeremy W. Dale, Malcolm von Schantz and Nick Plant. © 2012 John Wiley & Sons, Ltd. Figure 2.19 Tailing with terminal transferase.

From Genes to Genomes: Concepts and Applications of DNA Technology, Jeremy W. Dale, Malcolm von Schantz and Nick Plant. © 2012 John Wiley & Sons, Ltd. Figure 2.20 Cloning using homopolymer tailing.

From Genes to Genomes: Concepts and Applications of DNA Technology, Jeremy W. Dale, Malcolm von Schantz and Nick Plant. © 2012 John Wiley & Sons, Ltd. Figure 2.21 Cloning with a plasmid vector.

From Genes to Genomes: Concepts and Applications of DNA Technology, Jeremy W. Dale, Malcolm von Schantz and Nick Plant. © 2012 John Wiley & Sons, Ltd. Figure 2.22 Structure of the plasmid cloning vector pUC18.

From Genes to Genomes: Concepts and Applications of DNA Technology, Jeremy W. Dale, Malcolm von Schantz and Nick Plant. © 2012 John Wiley & Sons, Ltd. Figure 2.23 A recombinant plasmid formed using the cloning vector pUC18. The lacZ gene has been disrupted by insertion of a DNA fragment, resulting in white colonies on X-gal plates. bla, beta-lactamase (ampicillin resistance) – selective marker; ori, origin of replication; lacZ’, beta-galactosidase (partial gene); lacI, repressor of lac promoter.

From Genes to Genomes: Concepts and Applications of DNA Technology, Jeremy W. Dale, Malcolm von Schantz and Nick Plant. © 2012 John Wiley & Sons, Ltd. Figure 2.24 Bacteriophage growth: lytic cycle and lysogeny.

From Genes to Genomes: Concepts and Applications of DNA Technology, Jeremy W. Dale, Malcolm von Schantz and Nick Plant. © 2012 John Wiley & Sons, Ltd. Figure 2.25 Cohesive ends of lambda DNA.

From Genes to Genomes: Concepts and Applications of DNA Technology, Jeremy W. Dale, Malcolm von Schantz and Nick Plant. © 2012 John Wiley & Sons, Ltd. Figure 2.26 Replication of bacteriophage lambda DNA.

From Genes to Genomes: Concepts and Applications of DNA Technology, Jeremy W. Dale, Malcolm von Schantz and Nick Plant. © 2012 John Wiley & Sons, Ltd. Figure 2.27 Lambda insertion vector gt10.

From Genes to Genomes: Concepts and Applications of DNA Technology, Jeremy W. Dale, Malcolm von Schantz and Nick Plant. © 2012 John Wiley & Sons, Ltd. Figure 2.28 Use of lambda gt11 for generation of fusion proteins.

From Genes to Genomes: Concepts and Applications of DNA Technology, Jeremy W. Dale, Malcolm von Schantz and Nick Plant. © 2012 John Wiley & Sons, Ltd. Figure 2.29 Lambda replacement vector EMBL4.

From Genes to Genomes: Concepts and Applications of DNA Technology, Jeremy W. Dale, Malcolm von Schantz and Nick Plant. © 2012 John Wiley & Sons, Ltd. Figure 2.30 Structure of a cosmid.

From Genes to Genomes: Concepts and Applications of DNA Technology, Jeremy W. Dale, Malcolm von Schantz and Nick Plant. © 2012 John Wiley & Sons, Ltd. Figure 2.31 Structure and use of a yeast artificial chromosome vector.