lec 21b+22 - Lecture 21b + 22: Molecular techniques DNA...

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Lecture 21b + 22: Molecular techniques DNA cloning and DNA libraries
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Vector DNA fragment DNA cloning: general strategy -> to prepare large quantities of identical DNA Replication within host cells Recombinant DNA Isolation, sequencing, manipulation of purified DNA fragment (any piece of DNA derived from different sources) +
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Bacteria (Escherichia coli) Plasmids up to ~ 10 kb bacteriophages/Cosmids up to ~ 50 kb Bacterial artificial chromosomes (BACs) up to ~ 2Mb Yeast (Saccharomyces cerevisiae) Yeast artificial chromosomes Plasmids DNA cloning: Vectors
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DNA cloning: host cells: bacterial cultures agar plate individual E.coli colonies Petri dish Liquid culture Pick clone Grow bacteria Purify plasmid DNA from bacteria
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Cut and paste Restriction enzymes DNA ligases DNA cloning: Enzymes
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DNA cloning cut cut VECTOR (plasmid) OPEN VECTOR “INSERT” paste NEW RECOMBINANT DNA
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Restriction site = palindomic Single- stranded “tail” = overhang (can be 3’ or 5’) “Blunt” ends: ex. SmaI DNA cloning: Restriction enzymes GAATTC …CCCGGG… …GGGCCC… …CCC GGG… …GGG CCC…
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DNA cloning: Restriction enzymes 5’ overhang 5’ overhang 5’ overhang 3’ overhang 5’ overhang 3’ overhang 3’ overhang 5’ overhang 5’ overhang
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Restriction fragment EcoRI BamHI SmaI NotI ClaI BglII EcoRV SmaI XbaI XbaI XbaI All sites Unique sites BamHI EcoRV 145 159 180 221 DNA cloning: Restriction map Restriction digestion
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DNA cloning: Ligation ( ) ( )
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DNA cloning: Ligation !Blunt ->less efficient!
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Stripped from unessential sequences ->1.2-3 kb DNA cloning: Engineered E.coli plasmid
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DNA cloning: Introducing DNA in cells 1. Insertion in vector
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low efficiency of transformation! DNA cloning: Introducing DNA in cells
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lec 21b+22 - Lecture 21b + 22: Molecular techniques DNA...

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