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        Plasmid DNA Isolation from Bacteria

        互联网

        1222

        Materials:

        1.TENS solution:

        10 mM Tris (pH to 7.5)

        1 mM ethylenediaminetetraacetic acid (pH to 8.0 to dissolve)

        0.1 N sodium hydroxide

        0.5 % sodium dodecyl sulfate

        2.3 M Sodium acetate, pH 5.2

        3.Pre-chilled (at -20 ℃) 100 % ethanol

        4.70 % Ethanol

        5.Distilled water

        6.Overnight bacterial culture (LAB 5)

        Supplies:

        1.Micropipetter and tips

        2.Vortex mixer

        3.Microcentrifuge and tubes

        Procedures:

        1.Spin 1.5 ml of overnight bacterial culture for 30-60 seconds in a microcentrifuge. (observation: bacteria form a pellet at the bottom of the tube.)

        2.Decant supernatant, leaving 50-100 μl in the tube.

        3.Vortex to resuspend the bacteria pellet completely.

        4.Add 300 μl of TENS solution.

        5.Vortex for 5 seconds to mix. (observation: the contents of the tube should become slimy.)

        6.Add 150 μl of the sodium acetate.

        7.Vortex for 5 seconds to mix.

        8.Spin for 2 minutes in a microcentrifuge. (observation: a white pellet, containing bacterial debris, is formed at the bottom of the tube.)

        9.Transfer supernatant to a fresh tube.

        10.Add 0.9 ml of pre-chilled 100 % ethanol.

        11.Spin for 5 minutes in a microcentrifuge. (observation: a white pellet, containing plasmid DNA and bacterial RNA, is formed at the bottom of the tube.)

        12.Discard supernatant and add 1 ml of 70 % ethanol.

        13.Discard the ethanol and add another 1 ml of 70 % ethanol.

        14.Withdraw and discard as much liquid (ethanol) as possible then air-dry the pellet.

        15.Resuspend the pellet in 30 μl of distilled water and keep at 4 ℃or -20 ℃.

        Results:

        1.Plasmid DNA is now ready for estimation of DNA concentration (LAB 4) followed by restriction digest (LAB 3).

        2.Typical yield is 2-3 μg.

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