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    Dna07 2

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    Sabesan T
    1) Bacterial cells containing plasmid DNA are lysed using GET buffer, SDS/NaOH, and KOAc to release the plasmid DNA. 2) The mixture is centrifuged to separate the cell debris from the supernatant containing plasmid DNA. 3) Plasmid DNA is precipitated from the supernatant by adding ethanol and cooling, then collected by centrifugation into a pellet. 4) The pellet is dissolved in TE buffer to obtain a solution of purified plasmid DNA.

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    Dna07 2

    Uploaded by

    Sabesan T
    70 views2 pages
    1) Bacterial cells containing plasmid DNA are lysed using GET buffer, SDS/NaOH, and KOAc to release the plasmid DNA. 2) The mixture is centrifuged to separate the cell debris from the supernatant containing plasmid DNA. 3) Plasmid DNA is precipitated from the supernatant by adding ethanol and cooling, then collected by centrifugation into a pellet. 4) The pellet is dissolved in TE buffer to obtain a solution of purified plasmid DNA.

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    DNA07_2

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    1) Bacterial cells containing plasmid DNA are lysed using GET buffer, SDS/NaOH, and KOAc to release the plasmid DNA. 2) The mixture is centrifuged to separate the cell debris from the supernatant containing plasmid DNA. 3) Plasmid DNA is precipitated from the supernatant by adding ethanol and cooling, then collected by centrifugation into a pellet. 4) The pellet is dissolved in TE buffer to obtain a solution of purified plasmid DNA.

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    © All Rights Reserved
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    70 views2 pages

    Dna07 2

    Uploaded by

    Sabesan T
    1) Bacterial cells containing plasmid DNA are lysed using GET buffer, SDS/NaOH, and KOAc to release the plasmid DNA. 2) The mixture is centrifuged to separate the cell debris from the supernatant containing plasmid DNA. 3) Plasmid DNA is precipitated from the supernatant by adding ethanol and cooling, then collected by centrifugation into a pellet. 4) The pellet is dissolved in TE buffer to obtain a solution of purified plasmid DNA.

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    of 2

    Extraction of plasmid DNA from bacteria

    extraction of plasmid dna


    This is the classical method of isolating plasmid DNA; with it you can see the purpose of each step. Quicker,
    easier methods, using small resin filters that nest within a microcentrifuge tube, are also available as kits from
    suppliers of materials for molecular biology, but these tend to be too costly for everyday teaching purposes.
    Aim
    To isolate plasmid DNA from bacteria. The plasmids
    produced in this way can be cut and ligated in vitro and
    run on an electrophoresis gel. However, they should
    not be used for transformations as their identity cannot
    easily be verified.
    6.

    Preparation
    An overnight culture of E. coli containing a suitable (i.e.,
    self-cloned) plasmid is required. Sterile tubes and tips are
    not needed, however. Please see the safety note, below.

    Timing

    7.

    This activity takes about 60 minutes, including a period


    of 30 minutes when the materials are being chilled.

    8.

    Materials and equipment


    Needed by each person or group

    1 cm3 liquid culture of E. coli containing plasmid


    DNA, grown overnight at 37 C. It is best to use a
    plasmid with a high copy number (i.e., where
    numerous copies exist within each bacterial cell)
    such as pUC18 or pUC19. Antibiotic is needed in
    the medium to maintain the plasmid (e.g., ampicillin
    for pUC18, pUC19 or pBLU).
    Glucose / EDTA / Tris (GET) buffer, 100 L
    SDS / Sodium hydroxide (SDS / NaOH), 200 L
    Potassium acetate / Ethanoic acid (KOAc), 150 L
    Tris / EDTA (TE) buffer, 15 L
    Ice cold ethanol (95%), 400 L
    1.5 cm3 microcentrifuge tubes, 2
    Micropipettes or similar dispensing devices
    Tips for micropipette, 8
    Crushed ice, in an expanded polystyrene cup
    Microcentrifuge for 1.5 cm3 microcentrifuge tubes,
    capable of producing at least 8 000 x g. With a
    slower centrifuge (e.g., 2 0003 000 x g) the times
    stated here should be doubled.
    Hair drier

    Procedure
    1.
    2.
    3.
    4.

    5.

    Spin down 1 cm of bacterial culture in a 1.5 cm


    microcentrifuge tube for 1 minute or until a mass
    of cells some 23 mm in diameter is visible.
    Decant the supernatant (liquid) into disinfectant
    in a waste container.
    Ensure that all the liquid is removed, using a
    micropipette to draw off any that remains in the
    tube.
    Add 100 L of GET buffer to the tube. Resuspend
    the cells by closing the tube, then flicking the side
    repeatedly with a finger. EDTA in the buffer
    chelates Mg2+ and Ca2+ ions, which are necessary
    for DNase action and cell membrane stability.
    Add 200 L of SDS/NaOH solution. Mix by
    gently inverting the closed microcentrifuge tube.
    Leave to stand for 5 minutes at room temperature.

    26| ILLUMINATING DNA | Version 1.0 | June 2000

    9.
    10.

    11.
    12.

    13.

    SDS is an ionic detergent which dissolves the


    phospholipids and proteins of the cell membrane.
    This lyses the cells, releasing the DNA. The
    sodium hydroxide splits both the plasmid and
    chromosomal DNA into single-stranded
    molecules, but each denatured plasmid remains
    stuck as two intertwined rings.
    Add 150 L of KOAc and mix gently by inverting
    the closed microcentrifuge tube. Leave to stand
    on ice for 5 minutes. Treatment with KOAc
    restores the pH of the solution to neutral the
    DNA consequently renatures. KOAc also
    precipitates the SDS, phospholipids, proteins and
    large DNA molecules.
    Centrifuge the tube for 5 minutes to spin down
    the cell debris and chromosomal DNA.
    Very carefully transfer 400 L of the supernatant
    (liquid) to a clean microcentrifuge tube. Ensure
    that none of the cell debris is carried over. The
    supernatant contains the plasmid DNA.
    Add 400 L of ice cold ethanol. Mix gently.
    Leave the tube in a deep freeze (-1820 C) for
    1030 minutes, then centrifuge for 10 minutes at
    high speed. You should now have a very small
    but just visible pellet.
    Decant the supernatant into a waste container, taking
    care not to discard the pellet of plasmid DNA.
    With a micropipette, remove the last drops of
    liquid. If convenient, leave the uncapped tube to
    stand at room temperature for 15 minutes or use
    a hair drier so that all the alcohol evaporates.
    Dissolve the pellet in 15 L of TE buffer; vigorous
    mixing is needed to ensure that this happens. Store
    the plasmid solution in a freezer, or run it on a gel.

    Safety
    Handling microorganisms and ethanol in freezers
    Good microbiological practice should be observed when
    handling microorganisms. These procedures are
    explained on pages 1417. Please refer to the safety
    note about ethanol in freezers on page 20 also.

    resources
    Laborator y DNA
    Science. An
    introduction to
    recombinant DNA
    techniques and
    methods of genome
    analysis by Mark
    Bloom, Greg
    Freyer and David
    Micklos (1996) The
    Benjamin/Cummings
    Publishing Company,
    Menlo Park.
    ISBN: 0 8053 3040 2.

    running plasmid dna on a gel


    Procedure
    Wells
    1. Mix 15 L of plasmid DNA
    Relaxed circle
    solution with 4 L of loading dye.
    Plasmid
    2. Very carefully load the sample into DNA bands
    Linear
    a well in a 0.8% agarose gel.
    3. Run the gel until the loading dye
    Supercoiled
    RNA
    has moved 3040 mm.
    4. Pour off the buffer, leave the gel in Loading dye
    the tank and pour on 0.04%
    Azure A solution for 4 minutes only.
    5. Wash the gel surface with water.
    Relaxed: only one strand of the
    6. The plasmid bands will appear as
    plasmid is broken, hence the plasmid
    the stain moves down through the
    loses some of its coiling.
    gel. Three bands, corresponding to
    Linear: both strands of the DNA
    three physical forms of the plasmid,
    helix are broken.
    should be seen.
    Supercoiled: plasmid is tightly twisted.

    Spin 1 cm3 of bacterial culture for 1 minute


    at 8 000 x g, or until a pellet is formed.

    Pour the supernatant into disinfectant.


    Use a micropipette to remove as
    much liquid as possible from the pellet.

    Add 100 L of ice-cold GET buffer to the pellet.


    Cap the tube and resuspend the cells well by
    tapping the tube vigorously until no lumps remain.
    Glucose/EDTA/Tris

    Bacterial pellet
    23 mm diameter

    Glucose maintains the


    osmostic pressure and
    prevents lysis of the cells.

    Culture grown
    overnight at 37 C

    EDTA weakens the cell


    membrane and chelates
    Mg2+ ions that are needed
    by DNases, protecting
    the DNA from breakdown.

    Swirl before dispensing

    100 L

    Tris buffers the solution at


    pH8.

    Disinfectant

    1 cm3 of
    bacterial
    culture
    Remember to
    balance the centrifuge!

    Add 200 L of SDS + NaOH solution.


    Mix well by inverting the capped tube.
    Leave for 5 minutes on ice.

    Add 150 L of ice-cold KOAc solution.


    Mix well. A white precipitate should appear.
    Stand the tube on ice for 5 minutes.

    Spin down the cell debris for 5 minutes.


    The plasmid DNA remains in the supernatant.

    Potassium acetate + ethanoic acid

    200 L

    The ethanoic acid neutralises the


    solution, enabling the DNA
    to renature.The potassium
    acetate precipitates SDS,
    proteins, lipids and large
    DNA molecules.

    Remember
    to balance
    the centrifuge!

    150 L

    Cell suspension
    will become clear

    Plasmid DNA
    in supernatant

    Sodium Dodecyl Sulphate


    + Sodium hydroxide
    The SDS dissolves the
    cell membrane lipids and
    degrades the cellular proteins.

    Mix well
    by inverting
    the tube

    The NaOH splits the doublestranded DNA into single


    strands.

    Cell debris

    Transfer 400 L of the


    supernatant to a new tube.
    Add 400 L of ice-cold ethanol.

    10

    Leave the tube at -20 C for 1030 minutes.


    Centrifuge for 10 minutes at 8 000 x g.

    Remember
    balance the
    centrifuge ...

    400 L
    400 L

    11 13

    Decant the liquid from the tube.


    Remove any remaining liquid using
    a micropipette.

    Take care not to pour


    away or dislodge the
    very small DNA pellet
    Note:The pellet may
    almost be invisible.

    Plasmid
    suspension
    Leave the cell
    debris behind

    Mix gently
    by inverting
    the tube

    Plasmid DNA
    (and RNA)
    is insoluble in the
    ice-cold ethanol
    and precipitates.

    Dry the pellet thoroughly


    before dissolving it in 15 L
    of Tris/EDTA (TE) buffer.
    Store the plasmid solution
    in a freezer at -1820 C.

    www.ncbe.reading.ac.uk |27

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