Blunt ends and sticky ends “B| unt ends e.g. Sma I, Hae I, any other blunt-cutter CCCGGG GCC GGGCCC CCGG CCC CC CCCCC -GGG GG - GGGGG
Blunt ends and sticky ends • “Blunt ends” • e.g. Sma I, Hae III, any other blunt-cutter CCCGGG GGGCCC GGCC CCGG CCC GGG CC GG + CCCCC GGGGG
Plasmid: Cloning Vectors Origin of replication Selectable marker Cloning sites(unique) Shuttle vectors grow in more than one host cell
Plasmid: Cloning Vectors • Origin of replication • Selectable marker • Cloning sites (unique) • Shuttle vectors • grow in more than one host cell
Using plasmid vectors (a)Human DNA Plasmid vectors EcoRI site EcoRI site Q Origin of replication a are cut with Ecai ectors DNA and plasmid v Gene for ampicillin resistance Cleaved fragments and vectors are combined in the presence of ligase. Ligase (b)Recombinant plasmids are added to a population of E. coli cells Host chromosome 9os E coli plated onto medium containing ampicillin Cells containing recombinant plasmids are able to grow
Phage and cosmid vectors Foreign DNa Phage n chromosome fragment tithe tr gt fo eiazypna faplmcn 2 lika phag Incorporate into virus capsules 入R Infect E. coli host cells One bacterial cell Lawn of e. coli cells infected with recombinant viral dna Clear plaques contain many copies of single recombinant phage
Phage and cosmid vectors Phage A DNA Plasmid Incorporate into virus capsules Restriction cos sites amp site Or g n o 息息 replication Infect E. coli host cells Cleave dNA and plasmid with restriction enzyme. Splice cos DNA into plasmid cos sites F Cosmid vector Select for ampicillin resistance by growing infected cells on medium Linearize cosmid at restriction site containing ampicillin Combine with genomic DNA and ligate Genomic dna strands Surviving cells contain recombinant plasmids which may be cloned. Incorporate into virus capsules