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