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    E.Z.N.A. Endo-Free Plasmid Mini Kit II Spin Protocol

    互联网

    4480

    实验步骤

    Harvesting of Bacterial Cells:

    IMPORTANT: DO NOT EXCEED MAXIMUM CULTURE VOLUMES!

    High-copy-number maximum culture volume = 15 ml

    1. Harvest the bacterial cells: Pellet 10-15ml overnight culture by centrifugation. Transfer appropriate volume of culture to a 15 ml centrifuge tube and centrifuge at 5,000 x g for 10 minutes.

    Alkaline-SDS Lysis of Bacterial Cells:

    2. Decant or aspirate medium. To ensure that all traces of the medium are removed, use a clean paper towel to blot excess liquid from the wall of the vessel. To the bacterial pellet add 500 ul Solution I/RNase A. Resuspend cells completely by vortexing or pipetting up and down. Complete resuspension of cell pellet is vital for obtaining good yields.

    3. Transfer the sample to a 1.5 ml centrifuge tube. Add 500 ul Solution II and mix gently but throughly by inverting and rotating the tube 7-10 times to obtain a cleared lysate. This may require a 2-3 min incubation at room temperature with occasional mixing. Avoid vigorous mixing as this will shear chromosomal DNA and lower plasmid purity. (Store Solution II tightly capped when not in use.)

    4. Add 250 ul ice-cold Buffer N3 and mix gently but throughly by inverting tube several times until a flocculent white precipitate forms. Centrifuge at maxi speed (13,000 x g) for 10 minutes at room temperature.

    NOTE: The Buffers must be mixed throughly. If the mixture appears still viscous, brownish and conglobated, more mixing is required to completely neutralize the solution. Complete neutralization of the solution is vital of obtaining good yields.

    Endotoxin Removal using ETR Solution:

    5. CAREFULLY transfer the cleared supernatant to a clean 1.5 ml centrifuge tube. Add 0.1 volume of ice cold ETR Solution to the cleared lysate. Mix by inverting tube 7-10 times and incubate on ice for 10 minutes. Invert the tubeseveral times during the incubation.

    6. Incubate the lysate at 42℃ for 5 minutes. The lysate should appear turbid again. Centrifuge at maxi speed (13,000 x g) for 3 minutes at room temperature (22-25℃). The ETR Reagent should form a blue layer at bottom of the tube.

    7. Transfer the top aqueous phase (cleared lysate) into a new 2 ml microtube and add 0.5 volume of absolute ethanol (room temperature, 96-100%). Gently mix by inverting tube 6-7 times and incubate at room temperature for1-2 minutes.

    Plasmid DNA Purification with the HiBind® DNA Mini Column II:

    8. Equilibrate the HiBind® DNA column: Add 100ul Equilibration Buffer inro the column. Incubate at room temperature for 5 minutes. Spin at maximum speed (>13,000 x g) for 1 minute.

    9. Apply 750 ul of mixture from step 7 to the HiBind® Mini column II (purple) pre-inserted in a 2 ml collection tube, centrifuge at 10,000 x g for 1 minute at room temperature. Discard the flow-through and re-use the collection tube for next step.

    10. REPEAT step 9 by loading the remaining of the mixture into the same column until all of the mixture has passed through the column.

    11. Wash column with 500 ul Buffer HB and centrifuge as above. This step ensures that residual protein contamination is removed and must be included for downstream application requiring high quality DNA.

    12. Discard the flow-through liquid and wash the column by adding 700 ul DNA Wash Buffer diluted with ethanol. Centrifuge as above and discard flowthrough.

    NOTE: DNA Wash Buffer must be diluted with absolute ethanol before use. See label for directions. If refrigerated. DNA Wash Buffer must be brought to room temperature before use.

    13. REPEAT wash step 12 with another 700 ul DNA Wash Buffer diluted with ethanol.

    14. Discard the flow-through liquid. Centrifuge the empty column at maxi speed (13,000 x g) for 2 min to dry the column matrix. Do not skip this step-it is critical for removing ethanol from the column.

    Elution of Purified Plasmid DNA:

    15. Place the column into a new clean 1.5 ml micro-centrifuge tube. Add 60-100 ul (depending on desired concentration of final product) Endotoxin-Free Elution Buffer directly onto the column matrix and let it sit at room temperature for 2 minutes. Centrifuge at 13,000 x g for 1 min to elute DNA. This represents approximately 65-85% of bound DNA. An optional second elution will yield any residual DNA, though at a lower concentration.

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