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  • Re Comb Dna Competent Cell

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    Mohd Aiman
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    The document provides instructions for making competent E. coli cells for bacterial transformation. It involves growing E. coli in LB broth until mid-log phase, centrifuging the cells to form a pellet, resuspending the pellet in calcium chloride solution to make the cells competent, and storing the competent cells on ice until ready to use within 24 hours, without freezing, for bacterial transformation.

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    The document provides instructions for making competent E. coli cells for bacterial transformation. It involves growing E. coli in LB broth until mid-log phase, centrifuging the cells to form a pellet, resuspending the pellet in calcium chloride solution to make the cells competent, and storing the competent cells on ice until ready to use within 24 hours, without freezing, for bacterial transformation.

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    Attribution Non-Commercial (BY-NC)
    Download as PPT, PDF, TXT or read online from Scribd
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    Download as ppt, pdf, or txt
    15 views11 pages

    Re Comb Dna Competent Cell

    Uploaded by

    Mohd Aiman
    The document provides instructions for making competent E. coli cells for bacterial transformation. It involves growing E. coli in LB broth until mid-log phase, centrifuging the cells to form a pellet, resuspending the pellet in calcium chloride solution to make the cells competent, and storing the competent cells on ice until ready to use within 24 hours, without freezing, for bacterial transformation.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as PPT, PDF, TXT or read online from Scribd
    Flag for inappropriate content
    Download as ppt, pdf, or txt
    of 11

    Recombinant DNA Bacterial Transformation

    Student Instructions
    Making Competent Cells

    Recombinant Transformation
    Competent Cell Procedure
    Label a sterile 1.5 ml tube with CC and your groups name. From the Mid-Log suspension, transfer 1 ml of culture to your sterile 1.5 ml tube.

    Recombinant Transformation
    Competent Cell Procedure
    Place the tube in the microcentrifuge (MicroV microcentrifuge, shown) and spin for 2 minutes at 10,000 RPM.

    Recombinant Transformation
    Competent Cell Procedure

    Sterilely drain off the LB broth, leaving a visible pellet of bacteria in the tube.

    Recombinant Transformation
    Competent Cell Procedure

    Use a sterile plastic pipette to transfer 0.5 ml (10 drops) of calcium chloride to the tube containing the cells.

    Recombinant Transformation
    Competent Cell Procedure
    Resuspend the pelleted cell by vigorously fingerflicking (vortexing) the tube. Place on ice for 20 minutes.

    Recombinant Transformation
    Competent Cell Procedure

    After 20 minutes, place the tube in a microcentrifuge and spin for 2 minutes at 10,000 RPM.

    Recombinant Transformation
    Competent Cell Procedure

    Sterilely drain off the calcium chloride, being careful not to disturb the cell pellet.

    Recombinant Transformation
    Competent Cell Procedure

    Sterilely add 2 drops of fresh calcium chloride and finger vortex to resuspend.

    Recombinant Transformation
    Competent Cell Procedure
    Place cells suspended in calcium chloride on ice in the refrigerator (approx. 0oC) and store until you are ready to use them. DO NOT FREEZE.

    Recombinant Transformation
    Competent Cell Procedure
    Holding the cells at 0oC for 24 hours increases the cell competency.
    Cells can be held on wet ice (combination of melted and solid ice) for several days while you make the recombinant constructs.

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