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    (L3) - (NEET 2.0) - Biotechnology - 25th Sep

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    Omar Gill
    The document describes features of the cloning vector pUC8. It notes that pUC8 was developed to overcome limitations of pBR322 and has a high copy number. It contains the LacZ gene which allows identification of recombinant cells using blue-white screening on agar medium with ampicillin and the chromogenic substrate X-gal. White colonies indicate recombinant cells where the LacZ gene is inactivated by insert DNA.

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    (L3) - (NEET 2.0) - Biotechnology - 25th Sep

    Uploaded by

    Omar Gill
    111 views40 pages
    The document describes features of the cloning vector pUC8. It notes that pUC8 was developed to overcome limitations of pBR322 and has a high copy number. It contains the LacZ gene which allows identification of recombinant cells using blue-white screening on agar medium with ampicillin and the chromogenic substrate X-gal. White colonies indicate recombinant cells where the LacZ gene is inactivated by insert DNA.

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    [L3] - (NEET 2.0) - Biotechnology - 25th Sep

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    The document describes features of the cloning vector pUC8. It notes that pUC8 was developed to overcome limitations of pBR322 and has a high copy number. It contains the LacZ gene which allows identification of recombinant cells using blue-white screening on agar medium with ampicillin and the chromogenic substrate X-gal. White colonies indicate recombinant cells where the LacZ gene is inactivated by insert DNA.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    111 views40 pages

    (L3) - (NEET 2.0) - Biotechnology - 25th Sep

    Uploaded by

    Omar Gill
    The document describes features of the cloning vector pUC8. It notes that pUC8 was developed to overcome limitations of pBR322 and has a high copy number. It contains the LacZ gene which allows identification of recombinant cells using blue-white screening on agar medium with ampicillin and the chromogenic substrate X-gal. White colonies indicate recombinant cells where the LacZ gene is inactivated by insert DNA.

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    Cloning Vectors

    Biotechnology
    LECTURE 3
    Seep Pahuja
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    Cloning Vectors
    Biotechnology
    LECTURE 3
    Cloning Vector

    pUC8

    ➔ p = Plasmid
    ➔ UC = University of California
    ➔ It can be used to overcome the limitation of
    pBR322.
    Features of pUC8

    ➔ Copy number is high.


    ➔ It has Lac Z gene which codes for enzyme β-galactosidase .
    ➔ This enzymes cleaves lactose into glucose and galactose.
    ➔ Identification of recombinant cells achieved by a single step i.e., plating cells onto agar
    medium containing ampicillin and X-gal.
    ➔ X-gal is chromogenic substrate for β-galactosidase enzymes encoded by lac Z.
    Blue white Screening

    ➔ A recombinant DNA is inserted within the coding sequence of an enzymes, β-


    galactosidase.
    ➔ This results into inactivation of the gene for synthesis of this enzyme, which is referred to
    as insertional inactivation.
    ➔ The presence of a chromogenic substrate gives blue-coloured colonies if the plasmid in
    the bacteria does not have an insert.
    Screening of pUC8 recombinants

    Blue colony = non recombinant

    Agar + X-gal

    White colony = recombinant

    Blue colonies
    = β-galactosidase synthesized
    X-gal ➝ blue product

    White colonies
    = β-galactosidase not synthesized
    X-gal ➝ no blue product
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