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    Ayush Singh
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    The document describes various methods for introducing foreign DNA into host cells, including restriction enzyme digestion and ligation into plasmids, transfection using electroporation or lipoplexes, and microinjection of DNA-coated microcarriers. It also outlines common steps for extracting and purifying DNA from cells or tissues, such as cell lysis, protease and RNase treatment, and adsorption chromatography to isolate DNA. The goal is to generate recombinant cells containing and expressing the introduced DNA for research or therapeutic purposes.

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    The document describes various methods for introducing foreign DNA into host cells, including restriction enzyme digestion and ligation into plasmids, transfection using electroporation or lipoplexes, and microinjection of DNA-coated microcarriers. It also outlines common steps for extracting and purifying DNA from cells or tissues, such as cell lysis, protease and RNase treatment, and adsorption chromatography to isolate DNA. The goal is to generate recombinant cells containing and expressing the introduced DNA for research or therapeutic purposes.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Flag for inappropriate content
    12 views10 pages

    Biotech

    Uploaded by

    Ayush Singh
    The document describes various methods for introducing foreign DNA into host cells, including restriction enzyme digestion and ligation into plasmids, transfection using electroporation or lipoplexes, and microinjection of DNA-coated microcarriers. It also outlines common steps for extracting and purifying DNA from cells or tissues, such as cell lysis, protease and RNase treatment, and adsorption chromatography to isolate DNA. The goal is to generate recombinant cells containing and expressing the introduced DNA for research or therapeutic purposes.

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    © All Rights Reserved
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    of 10

    Origrin of mpliC'ahan

    IReslmfion
    ~nzyl[Lt ~i ~ti I

    Pol) IA)
    1

    R_~lric~j t11D

    sime,
    Site of cleavage

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    - and annealing

    Sticky ends
    ~~
    ~ y> Recombinant
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    :ampE;l
    p!BR322
    4
    SaJffl

    ~~ I ~
    E~ 001~· cl on Imrg ~ector pBR322
    Origin of
    replication

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    Antibiotic
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    lnser1ed
    gene

    Plasmid map

    marker

    Promoter
    Transcrip~on
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    LactJV
    prramotor
    ed e,w.e
    .,
    te:rml mat!IJrs

    EXPRESSION
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    Io + ~ -

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    +•-----+ -
    Electric field

    Cells and An electrical pulse Plasmids enter Pores seal with


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    mixed together pores to form the pores transformed cells
    Gell membrane Nucleus

    /
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    + endosome • .,. ,
    - -~ - - +
    + Llpoplex
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    • "l'\.,.
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    ~
    / +
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    +
    ~~
    ,r 7
    .---~'
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    ~ "'
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    lipid mixing

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    Lipopleit ~ t Endosomal
    +
    + + • Endosome maturation
    + +
    ~
    DNA
    fragmentation
    + +
    Lysosome

    Upoplex-mediated transfection and endocytosis


    Expert Reviews in Molecular MedicineC2003 Cambridge University Press
    M[cndn)aalon
    IIHHHll ai with
    tofldg., DNA,

    Haldl nu plpati

    Nuclll!!UI
    Helium gas
    -----------------------
    Gas cylinder

    Point for break

    Rupture disk

    c===:u.---- Plastic microcarrier


    DNA coated micropellets
    . i 1111 111 i i<
    · · • • · · · · · · -,:.~--------------- Stopping screen

    bObbOOb:b6·@) Target cell / tissue


    DNA extration

    Source

    Tissue extract
    Cell cult ure

    Cell Lysis
    Physical/Chemical
    Detergenv·Sep,aration of
    Wash steps DNA by specifi c adsorption
    1

    to membranes
    Removal of membrane lipids

    Wash steps J Prot ease treatment

    Protein denaturation and removal

    Wash steps I
    Removal of other cellular contaminants

    J RN ase t reat ment

    RNA removal
    Elute in slightly alkaline

    1 buffer/double distilled
    water

    Pu rified DNA

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