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    E.Z.N.A. Endo-Free BACs, PACs, P1s Protocol

    互联网

    1077

    实验步骤

     

    1. Isolate a single colony from a freshly streaked selective plate, and inoculate a starter culture of 2-5 ml LB or YT medium containing the appropriate selective antibiotic. Incubate for ~ 20-24 hr at 37°C with vigorous shaking (~ 300 rpm). Use a flask with a volume at least 4 times the volume of the culture.

    2. Pellet 1.5-5 ml bacteria by centrifugation at 13,000 x g for 3 min at room temperature. Decant or aspirate medium and discard.

    3. Resuspend the bacterial pellet by adding 200 ul of Buffer T1/RNase A solution, and vortexing. Complete resuspension of cell pellet is vital for obtaining good yields.

    4. Add 200 ul of Buffer T2 and gently mix by inverting 5-10 times to obtain a clear lysate. Incubate at room temperature for 5 minutes. Avoid vigorous mixing as this will shear chromosomal DNA and lower plasmid purity. The lysate should appear viscous. Do not allow the lysis reaction to proceed more than 5 min. (Store Buffer T2 tightly capped when not in use).

    5. Add 200 ul of Chilled Buffer T3 and gently mix by inverting 15-20 times until a flocculent white precipitate forms. Incubate on ice for 5 minutes. Do not vortex, as this will result in shearing.

    6. Centrifuge at $ 13,000 x g for 10 minutes at 4°C. Promptly proceed to the next step.

    7. Transfer the lysate into a new 2 ml microcentrifuge tube. Add 0.1 volume of ETR Solution (blue) to the filtered lysate, mix by inverting the tube 7-10 times and incubate on ice for 10 minutes. Inverting the tube serval times during the incubation.

    Note: After addition of ETR Solution, the lysate should appear turbid, but it should become clear after incubation on ice.

    8. Incubate the lysate at 42°C for 5 minutes. The lysate should appear turbid again. Centrifuge at 10,000 × g for 3 minutes at 25°C. The ETR Solution will form a blue layer at the bottom of tube.

    9. Carefully transfer the cleared supernatant to a new 1.5 ml tube (not supplied). Add 200 ul of BAC Binding Buffer diluted with isopropanol. Invert 3-5 times to mix throughly and incubate at room temperature for 5 min.

    Note: BAC Binding Buffer has to be diluted with isopropanol (96-100%) before use.

    See the label on the bottle or page 3 for detail instructions.

    10. Apply the sample to the HiBind® DNA column assembled in a 2 ml collection tube (provided).

    11. Centrifuge at > 8,000 x g for 30 second at room temperature. Remove the HiBind® DNA column, discard the flow-through and re-use the collection tube for next step.

    12. Place the HiBind® DNA column back into the collection tube and add 750ul of 80% ethanol. Centrifuge at > 8,000 x g for 30 seconds at room temperature. Discard the flow-through and re-use the collection tube.

    13. Place the HiBind® DNA column back into the collection tube and centrifuge at maximum speed for 2 minutes to dry the column.

    14. Place the HiBind® DNA column into a clean 1.5 ml centrifuge tube, apply 40 ul of Elution Buffer (10mM Tris-HCl, pH 8.5) or water onto the center of the membrane. Incubate 5 minutes at room temperature.

    15. Centrifuge at maximum speed for 2 minutes to elute the DNA.

    16. Store the eluted DNA at -20°C.

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