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    Plasmid Extraction Protocol

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    palsantanu8837276365
    The document describes a procedure for preparing plasmid DNA from bacterial cultures through alkaline lysis using SDS. Key steps include inoculating and growing a bacterial culture, lysing the cells with alkaline solutions, purifying the plasmid DNA through phenol-chloroform extraction and ethanol precipitation, and dissolving the purified plasmid DNA.

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    © All Rights Reserved
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    Plasmid Extraction Protocol

    Uploaded by

    palsantanu8837276365
    7 views3 pages
    The document describes a procedure for preparing plasmid DNA from bacterial cultures through alkaline lysis using SDS. Key steps include inoculating and growing a bacterial culture, lysing the cells with alkaline solutions, purifying the plasmid DNA through phenol-chloroform extraction and ethanol precipitation, and dissolving the purified plasmid DNA.

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    Plasmid extraction protocol

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    The document describes a procedure for preparing plasmid DNA from bacterial cultures through alkaline lysis using SDS. Key steps include inoculating and growing a bacterial culture, lysing the cells with alkaline solutions, purifying the plasmid DNA through phenol-chloroform extraction and ethanol precipitation, and dissolving the purified plasmid DNA.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    7 views3 pages

    Plasmid Extraction Protocol

    Uploaded by

    palsantanu8837276365
    The document describes a procedure for preparing plasmid DNA from bacterial cultures through alkaline lysis using SDS. Key steps include inoculating and growing a bacterial culture, lysing the cells with alkaline solutions, purifying the plasmid DNA through phenol-chloroform extraction and ethanol precipitation, and dissolving the purified plasmid DNA.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    of 3

    Preparation of Plasmid DNA by Alkaline Lysis with SDS: Minipreparation

    Principle: Plasmid DNA is isolated from small-scale (1-2 ml) bacterial cultures by
    treatment with alkali and SDS.

    Materials required: Alkaline lysis solution I, Alkaline lysis solution II, Alkaline
    lysis solution III, Antibiotic for plasmid selection, Ethanol, Phenol: Chloroform:
    Isopropyl alcohol (25:24:1, v/v), TE (pH 8.0) containing 20 µg/ml RNase A

    ❖ Alkaline Lysis Solution I (P1 buffer): [50 mM glucose, 25 mM Tris-Cl (pH 8.0),
    10 mM EDTA (pH 8.0)]

    • Prepare Solution I from standard stocks in batches of approx. 100 ml, autoclave
    for 15 minutes at 15 psi (1.05 kg/cm2) on liquid cycle, and store at 4°C.

    ❖ Alkaline Lysis Solution II (P2 buffer): [0.2 N NaOH (freshly diluted from a 10
    N stock), 1% (w/v) SDS]

    • Prepare Solution II fresh and use it at room temperature.

    ❖ Alkaline Lysis Solution III (P3 buffer): [5 M potassium acetate 60.0 ml, glacial
    acetic acid 11.5 ml, H2O 28.5 ml]

    • The resulting solution is 3M for potassium and 5M for acetate. Store the solution
    at 4°C and transfer it to an ice bucket before use.

    ❖ PCI reagent: Phenol: Chloroform: Isopropyl alcohol = 25:24:1


    Procedure:

    1. Inoculate 5 ml of rich LB Broth containing the appropriate antibiotic


    (ampicillin @100µg/ml) with a single colony of transformed bacteria.
    Incubate the culture overnight at 37°C with vigorous shaking @150 rpm.
    2. Pellet the bacterial cells in 1.5 ml microfuge tube by Centrifugation at
    maximum speed for 30 seconds at 4°C.
    3. Remove the medium by aspiration, leaving the bacterial pellet as dry as
    possible.
    4. Add 300 µl of P1 buffer (Resuspension buffer) and mix by vigorously
    vortexing the bacterial pellet with the solution.
    5. Add 300 µl of P2 buffer (Lysis buffer) to bacterial suspension. Close the tube
    tightly, and mix the contents by inverting the tube gently five times. Store
    the tube at room temperature for 5 minutes (Vortexing genomic DNA at this
    stage will shear DNA and contaminate purified plasmid DNA).
    6. Add 350 µl of ice-cold P3 buffer (Neutralization buffer). Close the tube and
    disperse the P3 buffer through the viscous bacterial lysate by gently inverting
    the tube several times. Store the tube on ice for 3-5 minutes.
    7. Centrifuge the bacterial lysate at maximum speed for 15 minutes at 4°C in a
    microfuge. Transfer the supernatant (approx. 700 µl to a fresh tube).
    8. Add an equal volume of PCI reagent. Mix the organic and aqueous phases
    by vortexing and then centrifuge the emulsion at maximum speed for 15
    minutes at 4°C in a microfuge. Transfer the aqueous upper layer of DNA to
    a fresh tube.
    9. Precipitate nucleic acids from the supernatant by adding 500 µl of 70%
    ethanol at room temperature. Mix the solution by vortexing and then allow
    the mixture to stand for 2 minutes at room temperature.
    10. Collect the precipitated nucleic acids by centrifugation at maximum speed
    for 10 minutes at 4°C in a microfuge. Remove the supernatant by gentle
    aspiration as described in Step 3 above.
    11. Repeat the step 10 to remove any beads of ethanol from sides of the tube.
    12. Store the open tube at room temperature until the ethanol has evaporated and
    no fluid is visible in the tube (5-10 minutes).
    13. Dissolve the nucleic acids in 20 µl of TE (pH 8.0) containing 20 µg/ml
    DNase-free RNase A (pancreatic RNase). Vortex the solution gently for a few
    seconds. Store the DNA solution at -20°C.

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