MIRADA Protocol for Filtering Water Samples

through 0.22 micron Sterivex Filters for DNA extraction

Assembly of Sterivex Filtration System*:

1. Clean area for Sterivex Filtration System setup. Attach PVC tubing to input connector of a 10L carboy and attach the outport to a vacuum pump. We like to put an in-line filter between the outport and the pump to prevent pump damage.
2. Wearing a clean set of gloves, carefully attach the sterile female/female luer-lock adaptors to the pipet-end in the stopper. Everything should be sterile from this point upward.
3. Label each sterivex filter accordingly. Then attach the male end of the filter to the top of the female adapter.
4. Attach the 140 cc syringe barrel to the female end of the sterivex (see photo on next page).
5. Quickly invert the 1L bottle of water onto the syringe barrel. (You may want to keep paper towels ready in case of spills but working quickly minimizes this in general).
6. After all three bottles are in place, turn on vacuum and open all red t-valves.
7. Let the filtration run until completion or the filter clogs. In the event that the later occurs, close the t-valve and remove the syringe barrel from the sterivex. Pour any remaining water into a graduated cylinder and record actual amount filtered.

Preparation of Sterivex**:

1. Reopen the t-value to continue filtration. Run the manifold until the sterivex filter is dry, then shut off red t-valve.
2. Remove sterivex filter and cap the bottom, or male end, with a blue end cap or female luer cap. Sometimes it is necessary to flick the sterivex with your wrist to get the final drops of liquid out before capping.
3. Add 2mL, or one pre-aliquotted tube, of Puregene Lysis Buffer to the Sterivex filter using a sterile transfer pipet. If buffer is not pre-aliquoted, it is better to filter the lysis buffer through a 0.22 um filter added through a syringe.
4. Cap the top or female end of the Sterivex with a blue end cap or a male luer cap.
5. Seal the connection at each end of the Sterivex filter with parafilm and place into a sterile Whirlpac bag.
6. These filters should be stored on dry ice for shipping purposes and frozen at –20 C if not shipped the same day. Lysis buffer is stable at 4C temp for short-term (samples will not be compromised if temporarily thawed).

* A second set of hands can aid in cleaning up spills from inverting the 1L bottle.

** Ideally, one person should handle the sterivex filter while a second person helps by opening blister packs for the caps and Lysis Buffer. This can be done by one individual with more difficulty but a bit of practice.

Sterivex Filtration Set-up: