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    E-Z 96® M13 Isolation Vacuum Manifold Protocol

    互联网

    1547

    实验试剂

     

    1. Sterile deionized water (or TE buffer)

    2. Absolute (96%-100%) ethanol

    实验设备

     

    1. Microcentrifuge capable of at least 10,000 x g

    2. Sterile 15 mL centrifuge tubes

    3. Sterile 1.7 mL centrifuge tubes

    4. Water bath preheated at 60°C

    实验步骤

     

    1. Grow the M13 infected bacteria in a 2.2 mL 96-well culture plate (not supplied).

    2. Spin down bacterial cells by centrifugation at 5000 rpm for 15 minutes at room temperature.

    3. Transfer 1-2 mL of the supernatant into a 2 mL Collection Plate (supplied). Be careful not to disturb the bacterial pellet during the transfer. If the supernatant is not clear, repeat the centrifugation step.

    4. Add 1/5 volume of MPG Buffer (200μl MPG per 1 mL culture) to the M13 supernatant and mix by vortexing. Incubate at room temperature for 15 minutes.

    5. Assemble the vacuum manifold by following the manufacturer’s instructions. If using Vac-03 vacuum manifold, place the waste collection tray inside the manifold and place the E-Z® 96 DNA Plate on top part of the manifold.

    6. Transfer 1 mL of the cleared supernatant from step 4 into each well of the E-Z® 96 DNA Plate. Switch on the vacuum for 2 minutes to draw the sample through. Note: Load 1mL of the sample at a time. If the volume of the sample is more than 1mL, ventilate the vacuum manifold and load another 1mL of the sample into each well of the E-Z® 96 DNA Plate.

    7. Add 1 mL of buffer MPX to each well of the E-Z 96® DNA Plate. Immediately apply the vacuum to draw all of the samples through the membrane.

    8. Stop the vacuum and add another 1 mL of buffer MPX to each well of the E-Z 96® DNA Plate. Incubate for 2 minutes at room temperature.

    9. Next, apply the vacuum until all of the liquid passes through the membrane.

    10. Add 1 mL of SPW Wash Buffer into each well of the E-Z 96® DNA Plate and switch on the vacuum until all of the liquid has passed through the membran.

    11. Remove the E-Z 96® DNA Plate from the vacuum manifold and remove any traces of liquid by tapping the E-Z® 96 DNA Plate firmLy on a stack of paper towels. Visually inspect the walls of the wells and make sure that all droplets are removed by tapping.

    12. Place the E-Z 96 DNA Plate back on the manifold and apply the vacuum for another 5 minutes. Repeat the tapping step from step 11 to completely remove any remaining liquid.

    13. Optional: Place the E-Z® 96 DNA Plate into a vacuum oven preset at 60°C and incubate for 10 minutes to completely dry the plate.

    14. Re-assemble the vacuum manifold by now replacing the waste collection tray with a 1.2 mL rack of microtubes (supplied). Place the E-Z® 96 DNA Plate on the top part of the manifold.

    15. Add 75-150ul of preheated (60°C) Elution Buffer (10mM Tris-HCl, pH 8.5) into the center of the membrane to each well of the E-Z® 96 DNA Plate. Incubate for 10 minutes. Apply the vacuum for 5 minutes to elute DNA. This represents approximately 75-80% of bound DNA. An optional second elution will yield any residual DNA, though at a lower concentration. The pH of the elution solution can also significantly affect the elution efficiency , therefore make sure that the pH of the water or TE is between 7.5-8.0.

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