TITLE: Collection and preservation of eDNA from marine water samples
AUTHORS: Ana Ramón-Laca, Abigail Wells, Linda Park

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left">This protocol is designed for water collection from Niskin bottles and filtration at sea using reusable filter cups.</div></div><div class = "text-block"><div class = "justify" style = "text-align:justify"></div><style>
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							</style></div><div class = "text-block"><div class = "justify" style = "text-align:left"><span style = "font-weight:bold;">Aim: to collect and filter 2.5 L of water at each depth from each CTD cast and preserved the filter at room temperature in lysis buffer</span></div></div><div class = "text-block"><div class = "justify" style = "text-align:justify"></div><style>
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[STEPS]
?. [Filter cups cleaning and assemblage]
Place all used small items in a small mesh bag and the filter cups in a large mesh bag in bleach for at least
?. [Filter cups cleaning and assemblage]
Rinse the abovementioned items for at least
?. [Filter cups cleaning and assemblage]
Allow to dry on the drying racks
?. [Filter cups cleaning and assemblage]
Wearing a fresh pair of gloves, mount filter cups with new sterile filter
?. [Collection of water from Niskin bottles]
Let the water from the hull (“surface water sample”) run for >  while the CTD is being deployed
?. [Collection of water from Niskin bottles]
Collect 2 samples ofeach of “surface water” for each CTD cast in 69oz whirlpack bags. Use pitchers for support and stability.
2.5 L
?. [Collection of water from Niskin bottles]
Once the CTD rosette has been brought back to the surface, water needs to be extracted from each Niskin bottle and transported into the lab for filtration.We want to collect of water from each Niskin.Wipe the spigot on the Niskin with DNAway to remove potential contaminants on the outside of the Niskin
2.5 L
?. [Collection of water from Niskin bottles]
Flush water (count to 10) from the Niskin to further reduce risk of contamination and collect L of water in a whirlpack bag and place in 2L pitcher
2.5 L
?. [Collection of water from Niskin bottles]
Include a sampling negative control by collecting  of distilled H2O (at least daily)
2.5 L
?. [Filtration of water samples]
Wipe working area with DNAway. Use a clean absorbant pad each day for the filtration station.
?. [Filtration of water samples]
Switch on the pump and set vacuum in the middle range between-8 and -12 bars (keep an eye on the gauge & adjust if necessary)
?. [Filtration of water samples]
Pour water from whirlpack bag (supported by pitcher) into the filter cup, repeat until finished (2.5L of water)
?. [Filtration of water samples]
Using DNA-free forceps fold filter and place in pre-labeled tube pre-filled with preservative buffer (, Longmire Buffer in a 5 ml LoBind tube).
2 mL
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?. [Filtration of water samples]
Filter  of ship distilled H2O every time the cleaning distilled H2O is replaced as a sampling negative control
2.5 L
?. [Filtration of water samples]
Every 2-3 days  of lab diH2O should be filtered as sampling negative controls
2 L
?. [Filtration of water samples]
Note tube label in notes. Also note time filtered, membrane type, place filtered (field/lab, which lab), etc.
?. [Filtration of water samples]
Store samples at room temperature away from UV light
?. [Filter cups cleaning and assemblage]
Set up wash station (two buckets, one with 0.5% bleach, one with clean distilled H2O)
Replace bleach and water stations at regular intervals (at least every other day, 0.5% bleach =   bleach in 20L bucket)