E-Z 96 Plant DNA Protocol-Vacuum Manifold Processing

Note: The following protocol is based on using OBI’s vacuum manifold (Product No. VAC-03).   1. Set up vacuum manifold by following manufacturer’s instructions.   2. Place waste collection tray inside the vacuum manifold, then place the E-Z 96 DNA plate on top part of the manifold.   3. Apply the entire sample (including any precipitate that may have formed) to an E-Z 96 DNA plate. Note: It is always good idea to mark the E-Z 96 DNA plate and collection plate at this stage so that they can be easily identified throughout the protocol. * Do not touch the rim of the wells with pipet tips to avoid crosscontamination.   4. Turn on the vacuum manifold and filter through the sample mixture by vacuum. Turn off the vacuum.   5. Add 700 ul DNA wash buffer into each well of the E-Z 96 DNA plate by using multichannel pipet. Turning on vacuum until all the liquid through the plate. (Dilute the DNA wash buffer with ETOH before use.)   6. Wash the plate with another 700 ul DNA wash buffer by repeating step 5.   7. Repeat Step 6 by washing the plate with 400 ul 100% ethanol. Continue vacuum until the E-Z 96 DNA plate is completely dried.   8. Remove the E-Z 96 DNA plate from manifold and tap hard on a stack of paper towels to remove any residue ethanol. Discard the flowthrough and collection plate.   Note: It is very important to completely dry the E-Z 96 DNA plate before elution. If a swing bucket centrifuge and 96-well plate adaptor are available, centrifuge at 4,000 x g for 10 minutes to dry the plate.   9. Assemble the manifold by placing a new 300 ul collection plate (supplied) inside the vacuum manifold. If an Omega VAC-03 is used, one 800 ul plate should be place under the 300 ul plate to give proper height for elution.   10 Place the E-Z 96 DNA plate atop the vacuum manifold.   11. To elute the DNA, add 100 ul of preheated (65°C) Elution Buffer to each well using a multichannel pipet. Incubate for 5 min at room temperature. Apply the vacuum to elute the DNA into collection plate.   TIP: 100 ul water or TE buffer is sufficient to elute up to 85% of the DNA from each well of the E-Z 96 DNA plate. A second elution step with same 100 ul elutate containing DNA, reheated to 65°C, will increase yield by up to 10-15%. Total DNA yields vary depending on type and amount of sample. Typically, 5-10ug DNA with a A260/A280 ratio of 1.7-1.9 can be isolated using 10 mg dried tissue.