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感谢你的观看 感谢你的观看 I genomic libraries and screening * 感谢你的观看 2109年5月18日 Genomic libraries and screening Genomic libraries cDNA libraries Screening procedure * 感谢你的观看 2109年5月18日 Genomic libraries Representative gene libraries Size of library Genomic DNA Vectors * 感谢你的观看 2109年5月18日 Representative gene libraries A gene library is a collection of different DNA sequences from an organism each of which has been cloned into a vector for ease of purification, storage and analysis. Type of gene library: the source of the DNA used. ? Genomic library ? cDNA library Representative library ? The library represents all the starting material, that is does it contain all the original sequences. ? cDNA libraries: lack some genes, enriched mRNA starting material * 感谢你的观看 2109年5月18日 Size of library It is possible to calculate the number (N) of recombinants (plaques or colonies) that must be in a gene library to give a particular probability of obtaining a given sequence. Formula: N＝ln(1-P)/ln(1-f) P: the desired probability; f : the fraction of the genome in one insert There are only a few thousand reconbinants whose insert size is 5-10kb to make good genomic libraries. For large genomes, the larger the insert size, the fewer recombinants are needed. (e.g. cosmid, YAC) * 感谢你的观看 2109年5月18日 For a probability of 0.99 with insert sizes of 20kb, these values for the E.coli (4.6*106bp) and human (3*109bp) genomes are: * 感谢你的观看 2109年5月18日 Genomic DNA To make a representative genomic library, genomic DNA must be purified and then broken randomly into fragments that are the correct size for cloning into the chosen vector. Genomic DNA purification Physical shearing and restriction enzyme digestion ? Physical shearing: pipeting, mixing or sonication Product: random fragments Most fragments have blunt ends. Cohesive ends: Klenow polymerase ? restriction enzyme digestion * 感谢你的观看 2109年5月18日 Genomic DNA