Course Overview
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Course Synopsis
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This course will enable students to understand the principle theories of recombinant DNA technology, advanced techniques for working with recombinant DNA, and the applications of the science being taught. This course is generally enlighten and familiarize the students to the hands-on lab practices, working with DNA, RNA by practicing molecular cloning and site- directed mutagenesis, measuring gene expression, and mutation analysis.
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Course Learning Outcomes
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Recombinant DNA Technology
- Learning outcomes of this course will be DNA extraction, RNA extraction, quantification of DNA and RNA, PCR nitty-gritties, gel electrophoresis, trouble shooting, recombinant DNA technology and techniques, gene and protein expression methods and techniques, restriction enzymes digestion, blotting techniques, transformation/transfection, electroporation, plasmid, plasmid extraction and expression systems
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Course Calendar
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Week 01
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2
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What is genetic engineering?
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4
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The flow of genetic information
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7
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Basic Techniques of Gene Manipulation-an overview
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8
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Isolation of genomic DNA
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9
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Agarose gel electrophoresis
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15
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Transformation of E. coli , Electroporation
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16
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Transformation with other organisms
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Week 02
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17
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Polymerase Chain Reaction (PCR)
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18
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Cutting and Joining DNA molecules
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19
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Host-controlled restriction modification
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20
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Types of restriction and modification (R-M) system
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21
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Nomenclature of restriction endonucleases
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22
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Target sites of restriction endonucleases
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23
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Number and size of restriction fragments
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24
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Summary of restriction endonucleases
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26
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Methods of joining DNA fragments
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27
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DNA ligase to create covalent recombinant DNA
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29
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Blunt end ligation via linker molecules
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32
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Cloning of cDNA by Homopolymer tailing
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Week 03
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34
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Interconversion of plasmid DNA
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35
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Effect of ethidium bromide on supercoiling of DNA
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36
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Phenotypic traits exhibited by plasmids
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37
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Properties of Conjugative and non-conjugative plasmids
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38
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Host range of plasmids
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39
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Partitioning and segregative stability of plasmids
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40
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Incompatibility of plasmids
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41
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Purification of plasmid DNA
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42
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Desirable properties of plasmid cloning vehicles
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43
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Natural plasmids as cloning vehicles
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44
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Use of pSC101 for cloning
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45
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pBR322, a purpose-built cloning vehicle
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47
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Improved vectors derived from pBR322
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48
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Runaway plasmid vectors
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Week 04
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49
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Bacteriophage lambda as cloning vector
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50
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Replication of phage-lambda DNA in lytic and lysogenic cycles
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51
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Modified lambda phages
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52
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Steps in cloning with lambda
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53
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Packaging phage-lambda DNA in vitro
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54
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Vectors for DNA sequencing: bacteriophage M13
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56
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Modified schemes for cloning in Cosmid vectors
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58
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Bacterial artificial chromosomes (BACs)
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59
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Yeast artificial chromosomes (YACs)
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60
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Shuttle and Expression vectors
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61
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Comparison of different cloning vectors-summary
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62
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Overview of cloning strategies
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64
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LambdaEMBL vectors for genomic library construction
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Week 05
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65
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Genomic libraries in high-capacity vectors
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66
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PCR as an alternative to genomic DNA cloning
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69
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Preparation of cDNA for cloning
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70
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Improved methods for cDNA cloning
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71
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PCR as an alternative for cDNA cloning
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73
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Screening by hybridization
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74
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Benton and Davis’ plaque lift procedure
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80
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Immunochemical screening
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Week 06
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81
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Immunochemical screening of lambdagt11
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82
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South-western and north-western blotting
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83
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Screening by functional complementation
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84
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Requirement for expression in E. coli
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87
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Stability of foreign proteins in E. coli
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88
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Constructing the optimal promoter
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89
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Optimizing translation initiation
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90
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Stability of mRNA and codon choice
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91
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The effect of plasmid copy number
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93
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Structural instability
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94
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Host cell physiology can affect the level of expression
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95
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DNA sequencing: Benefits and Applications
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Week 07
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97
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Chain termination or dideoxy procedure
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98
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Modifications of chain terminator sequencing
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99
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Automated DNA sequencing
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101
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DNA sequence databases
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104
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Primer extension: the single primer method
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105
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PCR methods for site-directed mutagenesis
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107
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PCR principles and procedure
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Week 08
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113
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Real-time quantitative PCR (qPCR)
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122
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PCR-Forensic sciences
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123
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PCR-Agricultural sciences and environment
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124
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PCR-Molecular paleontology
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126
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Markers for genome mapping
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127
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Genetic linkage mapping
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Week 09
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129
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Physical versus linkage maps
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130
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The use of RFLPs in physical maps
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132
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SNPs as physical markers
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133
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Polymorphic DNA detection in the absence of sequence information
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134
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AFLPs detection in the absence of sequence information
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135
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Fluorescence in situ hybridization (FISH)
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136
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Radiation hybrid (RH) mapping
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140
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High-throughput sequencing
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142
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Clone-by-clone sequencing
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143
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Orthologs and paralogs
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144
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Comparative genomics of bacteria
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Week 10
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145
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Comparative genomics of organelles
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146
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Comparative genomics of eukaryotes
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149
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Oligonucleotide Chips
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150
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Applications of Microarray-Microbial gene expression analysis
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151
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Applications of Microarray-Profiling in human disease
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153
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Screening phage display libraries
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154
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Applications of phage display
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155
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Knock Outs & Knock Inns
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157
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Applications of siRNA
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159
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Two-dimensional (2-D) electrophoresis
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Week 11
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162
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Cloning in gram –ve other than E. coli
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163
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Cloning in gram +ve bacteria B. subtilis
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164
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Cloning in Streptomyces
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166
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Cloning in S. cerevisiae
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167
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Transformation of fungi with exogenous DNA
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168
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Vectors for use in S. cerevisiae
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169
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Properties of different yeast vectors
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170
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Promoter system for yeast
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171
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Multipurpose vectors for use in yeast
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172
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Cloning of large DNA fragments
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173
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Deficiencies and advantages of YACs
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174
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Topic_174_Plant transformation
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175
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Topic_175_Callus cultrue
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176
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Topic_176_Cell suspension culture
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Week 12
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177
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Topic_177_Protoplast culture
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178
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Topic_178_Regenration of fertile plants
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179
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Topic_179_Major strategies for transformation
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180
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Topic_180_ Agrobacterium mediated transformation
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181
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Topic_181_T-DNA tansfer
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182
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Topic_182_Function of T-DNA gene transfer
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183
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Topic_183_Disarmed Ti vectors
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184
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Topic_184_Two plasmid strategy
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185
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Topic_185_Leaf disc transformation
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186
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Topic_186_Agrobacterium and monocots
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187
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Topic_187_Rhizogenes and Ri plasmids
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188
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Topic_188_Protoplast transformation
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189
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Topic_189_Particle bombardment
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190
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Topic_190_Chloroplast transformation
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191
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Topic_191_In planta transformation
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192
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Topic_192_Plant viruses as vectors
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Week 13
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193
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Topic_193_Genetic modification in animals
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194
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Topic_194_Strategies to transform animal cells
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195
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Topic_195_Chemical transfection techniques
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196
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Topic_196_Transfection with polyplexes
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197
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Topic_197_Transfection with liposomes and lipoplexes
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198
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Topic_198_Physical transfection techniques
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199
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Topic_199_Microinjections
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200
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Topic_200_Selectable markers for animal cell gene transfer
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201
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Topic_201_Bacterial vectors for animal transfection
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202
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Topic_202_Vrial vectors for transfection
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203
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Topic_203_Transgenic mice production
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204
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Topic_204_Pronuclear microinjection
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205
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Topic_205_Recombinant retroviruses
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206
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Topic_206_Transfection ES cells
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207
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Topic_207_Application of transgenic mice
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208
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Topic_208_Gene transfer to Xenopus
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209
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Topic_209_Gene transfer to Fish
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210
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Topic_210_Ways to exploit recombinant DNA
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Week 14
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211
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Topic_211_Production of recombinant proteins
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212
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Topic_212_Biopharmaceuticals approved
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213
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Topic_213_Transgenic animals and plants
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214
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Topic_214_Recombinant proteins in plants
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215
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Topic_215_Metabolic engineering in bacteria
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216
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Topic_216_Metabolic engineering in plants
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217
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Topic_217_Herbicide resistance in plants
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218
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Topic_218_Recombinant virus production
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219
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Topic_219_Fungal resistance
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220
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Topic_220_Resistance to bacteria
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221
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Topic_221_BT an insect resistance gene source
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222
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Topic_222_Drought resistance transgenic crops
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223
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Topic_223_Transgenic crops for poor quality soil
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224
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Topic_224_Biotechnology role in food
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225
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Topic_225_Transgenic model animals
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226
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Topic_226_Gene medicine
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227
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Topic_227_DNA vaccines
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228
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Topic_228_Gene augmentation therapy
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229
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Topic_229_Gene therapy in cancer
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230
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Topic_230_Ethics in gene manipulation
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