QIAGEN DNeasy Power Water SOP
Andrea Ottesen, Brandon Kocurek
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Abstract
QIAGEN DNeasy PowerWater SOP
Purpose: For the isolation of genomic DNA from filter water samples, inculding turbid water
Introduction: The DNeasy PowerWater Kit can isolate genomic DNA from a variety of filtered water samples. Utilizing Inhibitor Removal Technology® (IRT), even water containing heavy amounts of contaminants can be processed to provide DNA of high quality and yield. The DNeasy PowerWater Kit can isolate DNA equally well from any commonly used type of filter membrane. Purified DNA is ready to use in a final elution volume of 100 μl.
Principle & Procedure: The DNeasy PowerWater Kit starts with the filtration of a water sample onto a filter membrane. Filter membranes may be user supplied. The membrane is then added to our special 5 ml bead beating tube containing a unique bead mix. Rapid and thorough lysis occurs through vortexing in a specially formulated lysis buffer that enhances the isolation of microorganisms from filter membranes. After the protein and inhibitor removal steps, total genomic DNA is captured on an MB Spin Column. High-quality DNA is then washed and eluted from the MB Spin Column membrane for use in downstream applications including PCR and qPCR.
Before start
Solution PW1 must be warmed at 55°C for 5–10 minutes to dissolve precipitates prior to use. * Solution PW1 should be used while still warm.
- If Solution PW3 has precipitated, heat at 55°C for 5–10 minutes to dissolve precipitate.
- Shake to mix Solution PW4 before use.
- Perform all centrifugation steps at room temperature (15–25°C).
Steps
Procedure
Filter water samples using a filter funnel attached to a vacuum source. The volume of water filtered will depend on the microbial load and turbidity of the water sample. See below for types of water samples.
Clear water samples: Larger volumes of clear water can be processed because there is less chance of filter clogging.
Potable drinking water will generally allow for very high volumes depending on the quality and particulate count. In most cases, 100mL to 10L can be processed, although some users report processing even higher volumes.
Turbid Water Samples: Turbid samples with high levels of suspended solids or sediments will tend to clog filters with
smaller pore sizes (0.22 μm). Use of 0.45 μm filters is recommended for these types of samples. Prior to filtering, samples can be stored in a container to allow suspended solids to settle out. For samples where settling does not occur or is not desired, a method involving stacking filters with larger pore sizes on top of the filter membrane of the desired pore size is recommended. A common set-up is to stack a sterile 1 μm filter. This layering will filter out large debris and allow the smaller micron filter to trap microorganisms. The layered filter system can be washed with sterile water or sterile phosphate buffer to knock down some of the trapped microorganisms on the larger pore size filters. Although this is not completely efficient, it will increase the overall yield of microbial DNA.
If using a reusable filter funnel, remove the upper portion of the apparatus.
Using two sets of sterile forceps, pick up the white filter membrane at opposite edges and roll the filter into a cylinder with the top side facing inward.
Insert the filter into a 5mL PowerWater DNA Bead Tube.
Add 1mL of Solution PW1 to the PowerWater DNA Bead Tube.
For samples containing organisms that are difficult to lyse (e.g. fungi, algae) an additional heating step can be included. Heating can aid the lysis of some organisms (fungi, algae). After adding Solution PW1 (Step 5 of the protocol), heat the sample at 65°C for 0h 10m 0s . Resume protocol from step 6.
Secure the tube horizontally to a Vortex Adapter (cat. no. 13000-V1-5/13000-V1-15).
Vortex at maximum speed for 0h 5m 0s. Centrifuge the tubes at 4000x g (This centrifugation step is optional if a centrifuge with a 15mL tube rotor is not available, but will result in minor loss of supernatant).
Transfer the supernatant to a clean 2mL Collection Tube (provided). Draw up the supernatant using a 1mLpipette tip by placing it down into the beads. ( Note : Placing the pipette tip down into the beads is required. Pipette until you have removed all the supernatant. Expect to recover 600-650µL of supernatant.)
Centrifuge at 13000x g
Avoiding the pellet, transfer the supernatant to a clean 2mL Collection Tube (provided).
Add 200µL of Solution IRS and vortex briefly to mix. Incubate at 2-8°C for 0h 5m 0s .
Centrifuge the tubes at 13000x g .
Avoiding the pellet, transfer the supernatant to a clean 2 ml 2mL Collection Tube (provided).
Add 650µL of Solution PW3 and vortex briefly to mix.
Load 650 μl of supernatant onto an MB Spin Column. Centrifuge at 13000x g . Discard the flow-through. Repeat until all the supernatant has been processed.
Place the MB Spin Column Filter into a clean 2mL Collection Tube (provided).
Add 650µL of Solution PW4 (shake before use). Centrifuge at 13000x g .
Discard the flow-through and add 650µL of ethanol (provided) and centrifuge at 13000x g.
Discard the flow-through and centrifuge again at 13000x g.
Place the MB Spin Column into a clean 2mL Collection Tube (provided).
Add 100µL of Solution EB to the center of the white filter membrane.
Centrifuge at 13000x g.
Discard the MB Spin Column. The DNA is now ready for downstream applications.
QIAgen recommend storing DNA frozen ( -90°C to -15°C ) as Solution EB does not contain EDTA
