Generation of a library yields aa very large collection of plasmids each with a different chromosomal insert plasmid romosomal dna cut with restriction enzyme Amp mix and ligate Amp Amp transform by Cacl2 treatment select Amp Cloning by Complementation Say we wanted to clone the Lac operon. First a library would be made from dna from a Lac+ E coli strain. This library would then be used to transform a Lac" strain Transformants would first be selected by Ampr. The resistant colonies would then be screened for the ability to grow on lactose(Lac+). These clones should contain plasmids carrying a functional Lac operon How many clones would we need to screen? Each plasmid carries about 5 x 103 bp of chromosomal DNA. The chromosome is 5 x 106 base pairs so the entire genome will be covered if several thousand clones are screened All sorts of genes from E. coli have been cloned by looking for dna fragments that can restore function to a mutant. It is also possible to find genes from other bacteria. The following is a dramatic example of a cloning experiment to find an important protein for pathogenic bacteriumGeneration of a library yields a a very large collection of plasmids each with a different chromosomal insert. Amp E. coli r chromosomal DNA cut with restriction enzyme mix and ligate plasmid Ampr Ampr Ampr Ampr transform by CaCl2 treatment select Ampr Cloning by Complementation Say we wanted to clone the Lac operon. First a library would be made from DNA from a Lac+ E. coli strain. This library would then be used to transform a Lac– strain. Transformants would first be selected by Ampr. The resistant colonies would then be screened for the ability to grow on lactose (Lac+). These clones should contain plasmids carrying a functional Lac operon. How many clones would we need to screen? Each plasmid carries about 5 x 103 bp of chromosomal DNA. The chromosome is 5 x 106 base pairs so the entire genome will be covered if several thousand clones are screened. All sorts of genes from E. coli have been cloned by looking for DNA fragments that can restore function to a mutant. It is also possible to find genes from other bacteria. The following is a dramatic example of a cloning experiment to find an important protein for a pathogenic bacterium