lecture genecloning strategies for cloning · 2011. 9. 13. · strategies for cloning how to find...
TRANSCRIPT
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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
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Problem of selection!!
Fig. 8.1, Sid 127
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Different strategies for selection
Fig. 8.2, Sid 128
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Direct selektion
Fig. 8.3, Sid 129
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Direct selection for TrpA gene
Fig. 8.4, Sid 130
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Gene library in a cosmid
Fig. 8.5, Sid 132
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Expression of genes
Fig. 8.6, Sid 132
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Fig. 8.7, Sid 134
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Nucleic acid hybridization
Fig. 8.8, Sid 135
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Fig. 8.9, Sid 135
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32P-dATP
Fig. 8.10, Sid 136
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Labelling different techniques
Fig. 8.11, Sid 136
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Fig. 8.12, Sid 138
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Probing within a library to identifyan abundant clone
Fig. 8.13, Sid 139
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Fig. 8.14, Sid 139
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Fig. 8.15, Sid 140
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Fig. 8.16, Sid 141
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Heterologous probing
Fig. 8.17Sid 142
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Fig. 8.18, Sid 143
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Southern hybridization
Fig. 8.19, Sid 143
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Fig. 8.20Sid 144
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Immuno screening
Fig. 8.21, Sid 145
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PCR
• Design of primers• Different DNA polymerases• Error frequency of Taq Polymeras• Quantification of DNA and RNA
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Fig. 9.1 Sid 148
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Fig. 9.2, Sid 149
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Fig. 9.3, Sid 150
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Agarosegel electrophoresis
Fig. 9.4, Sid 150
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Amplification of α1-globin gene
Fig. 9.5, Sid 151
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short primers give unspecific binding
Fig. 9.6, Sid 151
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Typical temperature profile for PCR
Fig. 9.7, Sid 152
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Importance of correct annealingtemperature
Fig. 9.8, Sid 153
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Calculating Tm value for a primer
Fig. 9.9, Sid 154
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Fig. 9.10, Sid 155
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Combinatorial screening
Fig. 9.11, Sid 155
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Taq Polymeras add extra A
Fig. 9.13, Sid 156
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Fig. 9.14-9.15, Sid 157
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Error frequency of Taq polymeras
Fig. 9.16, Sid 158
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Quantification of PCR produkt
Fig. 9.17, Sid 159
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Real-time PCR
Fig. 9.18, Sid 160
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Quantification with Real-time PCR
Fig. 9.19, Sid 160
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Quantification of RNA
Fig. 9.20, Sid 161