Strategies for CloningHow to find the clone for a specific

gene?

Terms:• Direct selection• Different types of DNA libraries• Identification from a gene library• How to identify a clone

Problem of selection!!

Fig. 8.1, Sid 127

Different strategies for selection

Fig. 8.2, Sid 128

Direct selektion

Fig. 8.3, Sid 129

Direct selection for TrpA gene

Fig. 8.4, Sid 130

Gene library in a cosmid

Fig. 8.5, Sid 132

Expression of genes

Fig. 8.6, Sid 132

Fig. 8.7, Sid 134

Nucleic acid hybridization

Fig. 8.8, Sid 135

Fig. 8.9, Sid 135

32P-dATP

Fig. 8.10, Sid 136

Labelling different techniques

Fig. 8.11, Sid 136

Fig. 8.12, Sid 138

Probing within a library to identifyan abundant clone

Fig. 8.13, Sid 139

Fig. 8.14, Sid 139

Fig. 8.15, Sid 140

Fig. 8.16, Sid 141

Heterologous probing

Fig. 8.17Sid 142

Fig. 8.18, Sid 143

Southern hybridization

Fig. 8.19, Sid 143

Fig. 8.20Sid 144

Immuno screening

Fig. 8.21, Sid 145

PCR

• Design of primers• Different DNA polymerases• Error frequency of Taq Polymeras• Quantification of DNA and RNA

Fig. 9.1 Sid 148

Fig. 9.2, Sid 149

Fig. 9.3, Sid 150

Agarosegel electrophoresis

Fig. 9.4, Sid 150

Amplification of α1-globin gene

Fig. 9.5, Sid 151

short primers give unspecific binding

Fig. 9.6, Sid 151

Typical temperature profile for PCR

Fig. 9.7, Sid 152

Importance of correct annealingtemperature

Fig. 9.8, Sid 153

Calculating Tm value for a primer

Fig. 9.9, Sid 154

Fig. 9.10, Sid 155

Combinatorial screening

Fig. 9.11, Sid 155

Taq Polymeras add extra A

Fig. 9.13, Sid 156

Fig. 9.14-9.15, Sid 157

Error frequency of Taq polymeras

Fig. 9.16, Sid 158

Quantification of PCR produkt

Fig. 9.17, Sid 159

Real-time PCR

Fig. 9.18, Sid 160

Quantification with Real-time PCR

Fig. 9.19, Sid 160

Quantification of RNA

Fig. 9.20, Sid 161