实验试剂

Materials to Be Provided by User

1. Nuclease Free 1.5 and 2.0 mL Centrifuge Tubes

2. Ice

3. Isopropanol (100%)

实验设备

Materials to Be Provided by User

1. Tabletop with swinging-bucket rotor capable of 3,000 x g

2. Vacuum Manifold (Cat# Vac-03)

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℃ 600 with vigorous shaking until an OD of 2.0-4.0 is achieved.

2. Pellet 1-2 mL bacterial culture in a 2.0 ml 96-well deep-well plate by centrifugation at 3000 x g for 15 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 contains RNase A and lysozyme to each well of the plate.

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

5. Completely resuspend the cell pellet by vortexing the plate 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. Assemble the vacuum manifold according to the manufacturer’s instruction. For using Omega Bio-Tek’s Vac-03:

   1) Place E-Z 96 Xpress Plate on the top plate of manifold;

   2) Place a waste collection tray or 2 ml deep well plate inside the manifold base;

   3) Place the top plate of manifold over the base, the deep well plate or waste collection tray now should be positioned under the E-Z 96 X-press Plate. Seal the unused wells of E-Z 96 Xpress Plate with sealing film.

8. Transfer the entire cell lysate into E-Z 96 X-press Plate. Apply vacuum until all the lysate pass through the plate.

9. Apply the vacuum until all the lysate passes through the E-Z 96 Xpress Plate . Turn off the vacuum.

10. Add 500μl DLW Buffer (diluted with isopropanol) into Each well of E-Z 96 Xpress Plate.

11. Apply the vacuum until all the lysate passes through the Plate. After all the liquid pass through the plate, continue the vacuum for additional 3 minutes. Turn off the vacuum.

12. Remove the E-Z 96® DNA Plate from the vacuum manifold, then vigorously tap the plate on a stack of absorbent paper towels until no drops come out. Remove any residual moisture from the tip ends of the DNA plate with clean absorbent paper towels.

13. Place the E-Z 96 X-press Plate back to the vacuum manifold and apply the maximum vacuum for another 10 minutes. This step will evaporate any remaining ethanol from membrane.

14. Turn off the vacuum. Assemble the vacuum manifold by place a new 300 μl 96-well collection plate (provided) inside the base of manifold. If Omega manifold (Vac-03) is used in this procedure, a used E-Z 96 X-press Plate or a 800 μl plate should be placed under the 300 μl collection plate as a support to give the collection plate a proper position.

15. Add 100-150 μl Elution Buffer (10mM Tris-HCl, pH 8.5) or sterile water to each well of the E-Z 96® X-press Plate, let stand for 2 minutes. Apply maximum vacuum for 5-10 minutes to elute DNA from the plate. Turn off the vacuum and ventilate the manifold slowly.