The Vacuum Manifold is required for this procedure.
6. Connect the vacuum manifold to the vacuum source. Remove the manifold top and place a 96-well V-bottom sample plate and an DNA plate into the manifold base. Replace the manifold top and place an Filtration plate onto the manifold top. Be sure that filtration plate drip directors are aligned with the wells of the DNA plate underneath (Please refer to Figure 1).
Note: It is advisable to connect a trap between the vacuum manifold and the vacuum source to avoid contamination and damage to the vacuum pump or source.
7. Using a pipetter, transfer as much lysate as is possible from Step 5 to the corresponding wells of the Filtration plate. Turn on the vacuum source and adjust it to -25-30 inches Hg. Continue to draw vacuum through the plates until no liquid remains in any of the wells of the Filtration plate. Turn off the vacuum source and release the vacuum from inside of the vacuum manifold using the vacuum regulator. Discard the Filtration plate. Remove the DNA plate from the manifold and temporarily set it aside on a piece of absorbent toweling or plastic wrap. Note: Be careful when handling the DNA plate. It will contain a substantial volume of clarified lysate. Note: Wash and save V-bottom sample plate aside for next use. 8. Place the waste collection tray into the manifold base. Replace the manifold top. Place the DNA plate onto the manifold. Turn on the vacuum and adjust to –25-30 inches Hg. Continue to draw the vacuum until no liquid remains in any of the DNA plate wells. Leave the vacuum on.
9. Optional step: Add 0.5 ml of Buffer W1 to each well of the DNA plate. Continue to draw the vacuum until no Buffer W1 is present in any of the wells.
Note: The use of Buffer W1 is only necessary to remove traces of endonuclease when endA+ bacterial strains have been used for plasmid propagation. These include the JM series and HB101. Host strains, such as XL-1 Blue and DH5α do not require a Buffer W1 wash.
10. Add 0.9 ml of Buffer W2 to each well. Apply vacuum until no Buffer W2 remains in any of the plate wells. Repeat the Buffer W2 wash.
Note: Make sure that ethanol has been added to the Buffer W2 concentrate.
11. Apply maximum vacuum (up to –30 inches Hg) for 10 minutes to remove residual ethanol.
Note: Residual ethanol from Buffer W2 may inhibit subsequent enzymatic reactions and must be removed completely.
12. Turn off the vacuum source. Remove the DNA plate from the vacuum manifold and vigorously tap the plate approximately 6-8× on several layers of absorbent toweling. Be careful not to damage the drip directors on the underside of the plate. Note: We recommend the use of "lint-free" absorbent toweling to avoid the release of tiny fibers which could
contaminate the plasmid and interfere with subsequent capillary electrophoresis.
Note: Alternatively, the DNA plate can be centrifuged to remove residual Buffer W2. Please follow the following
procedure: Turn off the vacuum. Place the DNA plate onto a 1.6 ml growblock. Transfer to a centrifuge with a swinging bucket rotor and plate carriers. Centrifuge at ≥ 3,000×g for 5 minutes.
Note: The kit contains a limited number of 1.6 ml growblocks. Save and use in subsequent preparations.