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        E. Z. N.A.TM X-press Plasmid Protocol

        互联网

        1135

        实验试剂

         

        Materials to Be Provided by User

        1. Ice

        2. Isopropanol (100%)

        实验设备

         

        Materials to Be Provided by User

        1. Tabletop micro-centrifuge capable of 13,000 x g

        2. Nuclease Free 1.5 and 2.0 mL Centrifuge Tubes

        3. Vortex

        实验步骤

         

        1. Pick up a single colony from a fresh streaked selective plate and Inoculate 2-3 mL LB medium containing the appropriate selective antibiotic. Incubate 14-16 hours at 37℃ with vigorous shaking until an OD600 of 2.0-4.0 is achieved.

        2. Pellet 1-2 mL bacterial culture in a 2.0 ml centrifuge tube by centrifugation at maximum speed (13,000 × g) for 1 min at room temperature.

        Note: Do not use biomass large than 3.0. For example: If the OD600 of the culture is 2.0, use a 1.5 ml bacterial culture.

        3. Discard medium and remove any remaining liquid in the tube by using a pipettor or inverting the tube on an absorbent paper for 1 minute .

        4. Add 500μl ICE-COLD XCL Lysis Buffer which contains RNase A and lysozyme.

        Note: XCL Lysis Buffer must be ice cold to achieve maximum plasmid yield.

        5. Completely resuspend the cell pellet by vortexing at maximum speed for 30-60 seconds.

        Note: It is critical to fully resuspend the cell pellet to obtain optimized DNA yield.

        6. Incubate at room temperature for 3-5 minutes. The cell lysate should be nonviscous and slightly cloudy after incubation.

        7. Transfer the entire cell lysate into a X-press spin column inserted in a 2 ml collection tube.

        8. Centrifuge at maximum speed (13,000 x g) for 1 minute.

        9. Discard the flow-through liquid and re-use the collection tube.

        10. Add 500μl DLW Buffer (diluted with isopropanol) into the Xpress spin column.

        11. Centrifuge at maximum speed (13,000 x g) for 1 minute.

        12. Discard the flow-through liquid and re-use the collection tube.

        13. Place the empty X-press spin column back into the 2 ml collection tube. Centrifuge at maximum speed (13,000 x g) for 1 minute to dry the column.

        14. Transfer the X-press spin column into a new 1.5 ml centrifuge tube. Add 50 uL Elution Buffer (10mM Tris Hcl pH 8.5) directly onto the center of the membrane.

        15. Centrifuge at maximum speed (13,000 x g ) for 1 minute to elute the plasmid DNA.

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