Plasmids circular pieces ofâ€‌extrachromosomalâ€‌ DNA propagated inside host

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Plasmids circular pieces of”extrachromosomal” DNA propagated inside host have origin of replication -> ensures host will copy it. Plasmids circular pieces of”extrachromosomal” DNA propagated inside host have origin of replication -> ensures host will copy it Have selectable marker - PowerPoint PPT Presentation

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  • Plasmidscircular pieces ofextrachromosomal DNA propagated inside host have origin of replication-> ensures host will copy it

  • Plasmidscircular pieces ofextrachromosomal DNA propagated inside host have origin of replication-> ensures host will copy itHave selectable marker(usually a drug-resistance gene)-> ensures cell wont lose it

  • Plasmidscircular pieces ofextrachromosomal DNA propagated inside host have origin of replication-> ensures host will copy itHave selectable marker(usually a drug-resistance gene)-> ensures cell wont lose itpGLO also carries gene for Jellyfish Green Fluorescent Protein

  • Restriction Enzymesenzymes which cut DNA at specific sites

  • Restriction Enzymesenzymes which cut DNA at specific sitescalled "restriction enzymes" because restrict host range for certain bacteriophage

  • Restriction Enzymesenzymes which cut DNA at specific sitescalled "restriction enzymes" because restrict host range for certain bacteriophagebacterial immune system: destroy any non-self DNA

  • Restriction Enzymesbacterial immune system destroy non-selfDNA-> methylase recognizes same sequence in host DNA and protects it by methylating it

  • Restriction Enzymesenzymes which cut DNA at specific sitesUseful for fingerprinting: each cuts a particular DNA into a distinctive set of fragments

  • Restriction Enzymesenzymes which cut DNA at specific sitesUseful for fingerprinting: each cuts a particular DNA into a distinctive set of fragments

  • Restriction Enzymesenzymes which cut DNA at specific sitesUseful for fingerprinting: each cuts a particular DNA into a distinctive set of fragmentscan map each site by double digests

  • Restriction EnzymesRestriction enzymes create unpaired "sticky ends which anneal with any complementary sequence

  • Restriction EnzymesRestriction enzymescreate unpaired "sticky ends which anneal with any complementary sequencemake recombinant DNA by mixing 2 different fragments digested with same enzyme and gluing them together with ligase

  • Restriction EnzymesRestriction enzymescreate unpaired "sticky ends which anneal with any complementary sequencemake recombinant DNA by mixing 2 different fragments digested with same enzyme and gluing them together with ligaseGenetic Engineering

  • Restriction mapping

    Band

    distance

    traveled

    size

    1

    4

    21226

    2

    5.5

    "5000"

    3

    5.7

    4268

    4

    6

    3530

    5

    7.5

    2027

    6

    7.7

    1904

    7

    8.4

    1709

    8

    9

    1375

    9

    10.5

    947

    10

    11

    831

    11

    11.8

    564

  • Restriction mapping

    Band

    distance

    traveled

    size

    1

    4

    21226

    2

    5.5

    "5000"

    3

    5.7

    4268

    4

    6

    3530

    5

    7.5

    2027

    6

    7.7

    1904

    7

    8.4

    1709

    8

    9

    1375

    9

    10.5

    947

    10

    11

    831

    11

    11.8

    564

  • TransformationPhysical transfer of new DNA into cell1. Preincubation: chills cells, allows cations to neutralize negative charges on plasma membrane.

  • Transformation1. Preincubation: chills cells, allows cations to neutralize negative charges on plasma membrane.2. Incubation: allows DNA to diffuse through cell wall to contact plasma membrane.

  • Transformation2. Incubation: allows DNA to diffuse through cell wall to contact plasma membrane.3. Heat shock: opens holes in plasma membrane, allows plasmid to enter by diffusion.

  • Transformation3. Heat shock: opens holes in plasma membrane, allows plasmid to enter by diffusion.4. Recovery: cells must make proteins encoded by new DNA molecule before they can be plated.

  • Transformation5. Selecting transformants: cells which have taken up the plasmid are only ones which can grow in the presence of ampicillin.Form colony at place they landed on plate

  • DNA fingerprintingUse DNA sequences that vary between individuals

  • DNA fingerprintingUse DNA sequences that vary between individuals

  • DNA fingerprintingUse DNA sequences that vary between individualsMicrosatellitesshort sequences 2-5 bases long repeated multiple times

  • DNA fingerprintingMicrosatellitesshort sequences 2-5 bases long repeated multiple timesvary between families but not within families

  • DNA fingerprintingSTRs (simple tandem repeats)vary between families but not within familiesDetect by PCR with primers that bind outside repeat

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