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  • Cloning Flow Chart To Understand The Process

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    Ashwani Gautam
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    Flow sheet for cloning. A schematic representation of cloning in yeast. Select a vector, modification & expression

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    Cloning Flow Chart to Understand the Process

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    Flow sheet for cloning. A schematic representation of cloning in yeast. Select a vector, modification & expression

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as DOCX, PDF, TXT or read online from Scribd
    Flag for inappropriate content
    27 views1 page

    Cloning Flow Chart To Understand The Process

    Uploaded by

    Ashwani Gautam
    Flow sheet for cloning. A schematic representation of cloning in yeast. Select a vector, modification & expression

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as DOCX, PDF, TXT or read online from Scribd
    Flag for inappropriate content
    of 1

    Clone gene of interest into pA0815, pPIC3.5K, or pPIC9K.

    Create in vitro multimers using recombinant pA0815. Transform appropriate Pichia pastoris strain (GS115 for Mut+, KM71 for Muts).

    Plate transformants on histidine-deficient medium.

    Confirm Mut phenotype by testing on -his, +glucose and -his, + methanol plates.

    Screen transformants containing pPIC3.5K or pPIC9K constructs on various concentrations of G418. Select transformants with the highest reisistance to G418.

    Select 6-10 colonies of each Mut phenotype and test for expression.

    Select highest expressers for scale-up in a shake flask or fermentor.

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