MIRADA Protocol for
Filtering Water Samples
through 0.22 micron Sterivex Filters for DNA extraction
Assembly of Sterivex
- 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
- 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.
- Label each sterivex filter accordingly. Then
attach the male end of the filter to the top of the female adapter.
- Attach the 140 cc syringe barrel to the
female end of the sterivex (see photo on next page).
- 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).
- After all three bottles are in place, turn on
vacuum and open all red t-valves.
- 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.
- Reopen the t-value to continue
filtration. Run the manifold
until the sterivex filter is dry, then shut off red t-valve.
- 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
- 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.
- Cap the top or female end of the Sterivex
with a blue end cap or a male luer cap.
- Seal the connection at each end of the
Sterivex filter with parafilm and place into a sterile Whirlpac bag.
- These filters should be stored on dry ice for
shipping purposes and frozen at Ð20 C if not shipped the same day. Lysis buffer is stable at 4C temp
for short-term (samples will not be compromised if temporarily
* 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.