EZ 96 Plant DNA Protocol-Vacuum Manifold Processing-华强电子网

Note: The following protocol is based on using OBI's vacuum manifold (Product No. VAC-03).

1. Set up the vacuum manifold according to the manufacturer's instructions. Ensure all connections are secure and the system is leak-free before proceeding.

2. Place a waste collection tray inside the vacuum manifold, then position the EZ 96® DNA plate on top of the manifold. Make sure the plate is properly aligned for even vacuum application.

3. Apply the entire sample, including any precipitate that may have formed, onto the EZ 96® DNA plate. It’s important to ensure even distribution across all wells.

Note: At this stage, it's highly recommended to label both the EZ 96® DNA plate and the collection plate clearly to avoid confusion during the process.

* Avoid touching the rim of the wells with pipette tips to prevent cross-contamination between samples.

4. Turn on the vacuum manifold and allow the sample mixture to filter through under vacuum. Once the flow has stopped, turn off the vacuum.

5. Add 700 µL of DNA wash buffer into each well of the EZ 96® DNA plate. Before use, dilute the DNA wash buffer with ethanol as instructed.

6. Repeat step 5 by adding another 700 µL of DNA wash buffer to each well. This ensures thorough washing of the DNA bound to the plate.

7. Perform a final wash using 400 µL of 100% ethanol. Continue applying vacuum until the EZ 96® DNA plate is completely dry. Drying is crucial for optimal DNA recovery.

8. Remove the EZ 96® DNA plate from the manifold and tap it firmly on a stack of paper towels to remove any remaining ethanol residue. Discard the flowthrough and collection plate.

Note: It is very important to ensure the plate is fully dried before elution. If available, use a swing bucket centrifuge with a 96-well plate adaptor and centrifuge at 4,000 x g for 10 minutes to accelerate drying.

9. Reassemble the vacuum manifold by placing a new 300 µL collection plate (provided) inside. If using an Omega VAC-03, place an 800 µL plate underneath the 300 µL plate to achieve the correct height for elution.

10. Position the EZ 96® DNA plate on top of the vacuum manifold. Ensure it is centered and securely placed.

11. To elute the DNA, add 100 µL of preheated (65°C) Elution Buffer to each well using a multichannel pipette. Incubate for 5 minutes at room temperature. Then apply vacuum to collect the eluted DNA in the collection plate.

TIP: Using 100 µL of water or TE buffer is sufficient to recover up to 85% of the DNA from each well. For better yield, perform a second elution with the same volume of preheated elution buffer, which can increase DNA recovery by an additional 10–15%.

DNA yields may vary depending on the type and quantity of the starting material. Typically, 5–10 µg of DNA with an A260/A280 ratio of 1.7–1.9 can be isolated from 10 mg of dried tissue. Always verify purity and concentration using a spectrophotometer for accurate results.