TITLE: Collecting eDNA from marine water samples in the field
AUTHORS: Reindert Nijland

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the process of collecting eDNA from marine water samples in the field and four different approaches for preservation of filters with eDNA as well as eDNA extraction: </div><div class = "text-block"><ol style = "list-style-type: decimal;"><li style = "counter-reset:ol0;">Qiagen DNeasy kit</li><li style = "counter-reset:ol0;">Zymo DNA/RNA shield</li><li style = "counter-reset:ol0;">Longmire’s solution</li><li style = "counter-reset:ol0;">Alternative methods</li></ol></div></div>

[STEPS]
?. [Collection of eDNA from the water on a filter membrane]
Collect water in an appropriate sterile, DNA free container.
For marine samples collected during scuba diving, we use a small hand-pump and a punch-balloon.
?. [Collection of eDNA from the water on a filter membrane]
Filter - with the 250 ml filters (0.8 micron, cellulose nitrate) from Nalgene.
1 L
2 L
?. [Collection of eDNA from the water on a filter membrane]
Connect filter unit to vacuum pump, filter by vacuum.
I use a small vacuum-pump from ebay, powered with a 12V power pack. (DC12V Mini Vacuum Pump Negative Pressure Suction Pump)
?. [Collection of eDNA from the water on a filter membrane]
Using the vacuum, make sure the filter is dry, as to much transfer of liquid influences preservation and downstream DNA isolation.
?. [Collection of eDNA from the water on a filter membrane]
Cut filter from filter unit, it is glued in place. No problem to cut off outer 0.5 cm of filter. Use sterile scalpel + scalpel holder. Fold filter twice using scalpel+tweezers.
?. [Collection of eDNA from the water on a filter membrane]
Insert filter in 2 ml screwcap tube prefilled with .
[preservation solution]
It is possible to use other tubes, but not when using 3d-fuge, as these do not fit and will snap open during centrifugation.
?. [Collection of eDNA from the water on a filter membrane]
Shake tube with filter, to fully soak in the preservation solution. Repeat after - to re-soak. Filter ready for storage.
?. [Preservation of filters with eDNA]
The DNA is now collected on the filter. Either directly continue with the DNA isolation protocol of choice, or store the preserved the DNA on the filter, depending on preservation method used.Qiagen DNeasy kit, short term (Store filter in  from the DNeasy kit, shake well, store at  or below. Zymo DNA/RNA shield, short+longterm preservation:Store filter in , shake well, store at  or below.Longmire’s solution: longterm preservation:Store filter in -, shake well, store at RT or below
[ ATL buffer]
0 Room temperature
[DNA/RNA Shield]
0 Room temperature
500 µl
[longmire’s solution]
Other alternative preservation methods: ethanol, freezing, DMSO, DESS, dehydration, combinations, etc.
?. [eDNA extraction]
When in the lab, add .
[protK]
?. [eDNA extraction]
Follow standard protocol for blood samples: Since using  volume, double amount of AL and Ethanol: add . Mix thoroughly by vortexing, add . mix by vortexing, load on spin column, etc..
400 µl
[Buffer AL]
[Ethanol]
Pipetting is a bit of hassle with the filter in the tube, best to remove most buffer, spin shortly, remove rest. Also you will need to load the DNA filter tube twice to process all liquid. Elute in either recommended   or use lower elution volume (down to  ) if more concentrated DNA is required.
?. [eDNA extraction]
Add .
[Buffer AL]
?. [eDNA extraction]
Mix thoroughly by vortexing.
?. [eDNA extraction]
Add .
[Ethanol]
?. [eDNA extraction]
Mix by vortexing.
?. [eDNA extraction]
Load on spin column.
?. [eDNA extraction]
In the lab, add .
[ProtK]
?. [eDNA extraction]
Incubate - at .
55 °C
?. [eDNA extraction]
Incubate at  for -.
56 °C
?. [eDNA extraction]
Add 1 volume of genomic extraction buffer, follow protocol (mix by vortexing, load on spin column, etc.).
Pipetting is a bit of hassle with the filter in the tube, best to remove most buffer, spin shortly, remove rest.Elute in either recommended volume ( ) or use higher elution volume if more dilute DNA is required.
?. [eDNA extraction]
Before doing DNA extraction incubate for  with protK at .
Use classic phenol/chlorophorm extraction, that works also very well. I did this with the samples I preserved in Longmire solution, can also be done with samples in DNA/RNA shield.
56 °C
?. [eDNA extraction]
Either use a 15ml tube, or the following idea will also work: cut the filter from the 500 ml units in 4 equal pieces and process these separately. Isolate DNA from 2 quarters, store the other 2 in Longmire for later if something goes wrong with the isolation.
With the larger filter units (500ml) you also get larger filters, which do not fit in a 2ml tube.
?. [Isolation of eDNA from Longmire preserved filter-membranes]
If filters in 2ml tubes and volume
Optional when using 15ml tubes: Add several sterile, DNA free, 4mm glass-beads.
?. [Isolation of eDNA from Longmire preserved filter-membranes]
Add .
[proteaseK ]
?. [Isolation of eDNA from Longmire preserved filter-membranes]
Vigorously vortex the tube with filter+longmire solution.
?. [Isolation of eDNA from Longmire preserved filter-membranes]
Incubate for  –  at -, with regular vortexing.
56 °C
60 °C
?. [Isolation of eDNA from Longmire preserved filter-membranes]
Add 1 volume of Phenol/Chlorphorm/Isoamylalcohol (PCI) (-usually).
2 mL
3 mL
?. [Isolation of eDNA from Longmire preserved filter-membranes]
Homogenize on vortex mixer or vortex for ~.
?. [Isolation of eDNA from Longmire preserved filter-membranes]
Spin down  at .
?. [Isolation of eDNA from Longmire preserved filter-membranes]
Collect waterphase.
Waterphase now pink or more dark red (some samples). PCI yellowish, but not pink.
?. [Isolation of eDNA from Longmire preserved filter-membranes]
Second extraction with -. Spin for  at .
2 mL
[Chlorophorm/IAA]
?. [Isolation of eDNA from Longmire preserved filter-membranes]
Collect ~- of the waterphase.
2 mL
3 mL
?. [Isolation of eDNA from Longmire preserved filter-membranes]
Add .
[NaAcetate 3M]
?. [Isolation of eDNA from Longmire preserved filter-membranes]
Precipitate  at .
-20 °C
?. [Isolation of eDNA from Longmire preserved filter-membranes]
Invert some more for about .
Often a red-pinkish colour develops (probably some reaction between the algal chlorophyll and the PCI?)
?. [Isolation of eDNA from Longmire preserved filter-membranes]
Add .
[cold EtOH]
White precipitate forms, this is normal.
?. [Isolation of eDNA from Longmire preserved filter-membranes]
Spin down for  at .
Sometimes yellowish sticky/slimy pellet visible(polysaccharides?), can be removed by washing with 70% EtOH. Necessary to resuspend the gooey pellet by shaking/vortexing.
?. [Isolation of eDNA from Longmire preserved filter-membranes]
Wash pellet in . (1/2)
[70% EtOH]
?. [Isolation of eDNA from Longmire preserved filter-membranes]
Wash pellet in . (2/2)
[70% EtOH]
?. [Isolation of eDNA from Longmire preserved filter-membranes]
Dry pellet to air.
?. [Isolation of eDNA from Longmire preserved filter-membranes]
Resuspend in -.
100 µl
[NFW/T10E1/EB]