Cloning Vectors

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Recombinant DNA

• Cloning Vectors
• plasmids
• viruses/phage
• lambda (?)
• filamentous/M13
• (ssDNA, display)
• combinations

• Major Limitation of Plasmids
• transformation is inefficient
• 105-109 colonies per mg DNA for heat shock
• 109 -1010 colonies per mg DNA for electroporation
• 1 mg of a 5 kb plasmid 2 x 1011 copies
• efficiency ? with ? size (10-20 kb maximum)

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Bacteriophage ?

• large phage that infects E. coli
• phage attaches to bacteria and injects DNA
• complex genetics and life cycle with two phases
• lytic
• lysogeny

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? as a Cloning Vector

• infectious ? can be assembled in vitro
• foreign DNA can be incorporated into the ? genome
  
• non-essential genes removed
• phage assembly can occur with 40-52 kb of DNA
  (wild-type ? ? 50kb)

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Cloning in ?

• Target DNA
• digest gDNA
• make cDNA
• Vector DNA
• purchase pre-cut and dephosphorylated arms
• add adapters if needed

• Prepare foreign DNA
• Prepare vector DNA
• Mix vector, foreign DNA and ligase
• In vitro packaging
• Infect host E. coli
• Screen plaques
• Plaque purification
• Subclone fragment into plasmid

EcoRI BamHI AATTCGAACCCCTTCG
GATCCNNNNNN---- GCTTGGGGAAGCCTAG
GNNNNNN----
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Cloning in ?

• Prepare foreign DNA
• Prepare vector DNA
• Mix vector, foreign DNA and ligase
• In vitro packaging
• Infect host E. coli
• Screen plaques
• Plaque purification
• Subclone fragment into plasmid

COSLLLLLLLLG AATTCFFFFFFFG AATTCRRRRRRRRR

LLLLLLLLCTTAA GFFFFFFFCTTAA GRRRRRRRRRCOS
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In Vitro Packaging

• rDNA packaging extract
• phage particles self assemble

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Cloning in ?

• Prepare foreign DNA
• Prepare vector DNA
• Mix vector, foreign DNA and ligase
• In vitro packaging
• Infect host E. coli
• Screen plaques
• Plaque purification
• Subclone fragment into plasmid

Library mixture of phage with different DNA
inserts
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• Cloning in l
• Prepare foreign DNA
• Prepare vector DNA
• Ligation reaction
• In vitro packaging
• Infect host E. coli (plate on lawn)
• Screen plaques
• Plaque purification
• Subclone fragment into plasmid

Plaque clear zone on bacterial 'lawn' cause by
lytic phage
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Plaque Lift
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• Cloning in l
• Prepare foreign DNA
• Prepare vector DNA
• Ligation reaction
• In vitro packaging
• Infect host E. coli (plate on lawn)
• Screen plaques
• Plaque purification
• Subclone fragment into plasmid

• amplify cloned phage
• purify phage DNA
• excise insert
• ligate into plasmid

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Identifying Recombinants

• based on interruption of a gene
• eg., lacZ gene b-galactosidase
• intact b-galactosidase produces blue color in
  presence of X-gal
• ?-complementation or blue-white screening

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Probes Based on Protein

• mixed synthetic oligonucleotides based on partial
  protein sequence
• antibodies

A-T-G-T-G-G-G-A-R-Y-T-N-A-T-H-G-C-N-G-G-N 1x1x1x1x
1x1x1x1x2x2x1x4x1x1x3x1x1x4x1x1x4 768
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Screening Expression Libraries

• prepare library in expression vector
• cDNA better than gDNA
• (introns and reading frames)
• screen with protein based probe
• antibody/western blot
• ligand binding or other activity

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