DeWalch DNA Purification System
(NeXPrep Kit Protocol Release 2.0)

Kit Materials:  DeWalch 96 well NeXplate, collection microtiter plate, Solution A (Lysis Buffer), Solution B (50% IPA Wash Buffer), and Solution C (80% IPA Wash Buffer).
Materials not supplied:  Bacterial growth media, Isopropanol (IPA), and molecular grade water
Equipment: DeWalch Vacuum Manifold, DeWalch 96 well piercer, microtiter plate centrifuge

Prior to starting procedure:

1) Prepare wash buffer

Wash Buffer:  Prior to beginning procedure add Isopropanol to the fill line of both the 50% Wash Buffer (~200 ml of IPA) and the 80% Wash Buffer (~160 ml of IPA ).  Be sure to check and date the filled box on the bottle.

2) Assemble pipettes, tips and vortexer.
3) Set up vacuum manifold and piercer
4) Prepare overnight Culture.  1ml Cultures may be grown in the NeXplate. Do not remove the DNA binding matrix, which is compatible with bacterial growth.

Opening plates: Holding the plate upright, rap the block sharply on a flat table surface prior to peeling the foil covering.  Each well is packed with powdered binding media.

IF CELLS ARE GROWN IN A SEPARATE 96 WELL BLOCK, TRANSFER TO THE DEWALCH BLOCK PRIOR TO STARTING PROCEDURE

Detailed Procedure:

1.        Harvest cells in the NexPlate by centrifugation of 1ml of overnight cultures at 2500g for 5 minutes.  Carefully pour off the supernatant.

2.        Lyse cells by the addition of 200ml of Solution A (Lysis Buffer) to each well and vortex vigorously for 30 seconds.  Incubate at room temperature for 1 minute. Use caution as the lysis buffer contains chaotropic salts 

3.        Pierce bottom of the NeXplate in the DeWalch 96 well piercer:  Place the NeXplate in the piercer, making sure the nozzles of the block are aligned correctly with the piercing pin array.  Pull the lever over to the down position and press. This pierces the NeXplate.  Return the lever to the up position and remove the pierced block.  Place the block on the DeWalch vacuum manifold. DO NOT apply vacuum yet

4.        Add 400 ml of Solution B (50% IPA Wash Buffer) to each well. (Do Not Mix!)  Incubate at room temperature for 1 minute.        

5.        Evacuate Lysis and Wash Buffers using the vacuum manifold.  Evacuate for 30 seconds or until all the liquid has been removed from the wells.  

6.        Add an additional 400ml of Solution B to each well.  Incubate at room temperature for 2 minutes.  Evacuate Solution for approximately 30 seconds using the vacuum manifold. 

7.        Add 400ml of Solution C (80% IPA Wash Buffer) to each well. Evacuate Wash Buffer using the vacuum manifold. Continue applying vacuum for 10 - 12 minutes or until the wells are completely dry.  Remove the Block from the vacuum manifold.

8.        Place a microtiter plate on the vacuum manifold and place the Block on top of the microtiter plate making sure the nozzles of the block are lined up and resting in the chimneys of the microtiter plate.

9.        Elute the DNA by the addition of 120ml of molecular grade water.  Incubate at room temperature for 3 minutes and elute for 10 - 30 seconds using the vacuum manifold.  (Do not elute for long periods of time.) 

10.     Collect the liquid in the bottom of the microtiter plate with a quick spin (low speed centrifugation). 

DNA IS NOW READY FOR USE