TITLE: Water Sampling onto Filters for Nucleic Acids Sequencing
AUTHORS: Melissa Duhaime, Morgan Lindback, Rachel Cable

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol will guide you through the process of filter water using a serial filtration approach to generate filters with microbial biomass. Later, nucleic acids will be extracted from these filters.</div></div>

[STEPS]
?. [Assembly of pump and filtration units]
The goal of this step is to prepare unit such that direction of flow in tubing will be from water source, through pump head, through first filtration unit, through second filteration unit, and then into collection receptacle. Attach tubing to both ends of a 47 mm filter unit.Run tubing through pump head.Dissable unit and leave the downstream part detached (the half on which the filter will be placed).Pump approx. 200 ml of the Place a 3 µm filter into the filter unit and close it. Ensure that all O-rings are in place or the filter unit will leak when under pressure...losing your precious microbes.Now the second filtration unit will be attached to the first. Disassemble the second filtration unit, such that only the top half is connected to the system. Again, pump approx. 200 ml of the Replace the 3 µm filter (save this first filter for later itn he day when you need to clean the set-up again). Place a 0.22 µm filter into the second filter unit and close it.  Now the system is set-up as described above: water source, pump head, first filtration unit, and second filteration unit. All the tubing has been flushed with station water of the approriate size fraction.
 Note: If you were interested in collecting viruses, you would collect the
?. [Collect water]
Pre-filter the water with a 20 µm mesh net and fill a clean carboy with several liters of this
?. [Pumping the water (perform this in triplicate for each sample)]
Pump water through the system at a moderate rate. You want to avoid high pressure in the system, so that things don't push through that would not naturally push the filters.Collect the flow through in a waste collection bottle with graduated markings (or a hand-drawn line indicating 1 L volume). We measure the water collected in this vessel to know how much water has passed through the filters.Pump 1 liter of water through. Stop the pump. Remove the 3 µm and 0.22 µm filters using sterile tweezers. Place the filters inside a cryovial. It is helpful to use two pairs of tweezers to roll or fold the filter. Shoving it into the cryovial is also acceptable if neccessary (e.g., running from a storm), but wrinking it will annoy you later when you do the DNA extractions.Drop cryovial with filter into liquid nitrogen (LN2).If the collection rate slows markedly or the pump appears to be building up high pressure in the system before 1 L has been fitlered, the filters (most likely the 3 µm) may be clogged. If this happens, stop the pump and replace the filters. You may use more than one filter if needed to get through 1 full liter of water. Filters can be conbined in the downstream processing. Be sure to note this process. It is useful to first start by replacing the 3 µm, as that is typically what limits the rate.
?. [Cleaning the pump]
Optimally, we clean the tubing and filtration units between all stations. If this is not possible logistically, flush with station water at each new station (described above).Pump a small amount(ca. 0.50 L) of 10% bleach through the tubing and assembled filtration units (with not filters in place). Collect the outgoing bleach, as it can be reused to clean the pump in the future.After the 10% bleach has been fully pumped through, pump another ca. 1-2 L of Deionized water (or MilliQ) through the pump to flush any bleach that remains. You know you are done flushing with water when the system no longer smells of bleach.