TITLE: DNA extraction from environmental size-fractionned plankton samples
AUTHORS: Sarah Romac

[DESCRIPTION]
This protocol has been designed for total DNA extraction from environmental marine water samples. Samples from different plankton size fractions are collected by Niskin bottles (Step Case 1) or by plankton net tows (Step Case 2) and are filtered on polycarbonate 47mm filters.

This procedure works for metabarcoding and metagenomics using short-read NGS (Illumina) but also works for long-read metabarcoding (Oxford Nanopore Technologies). 

This protocol is applied in routine in our lab.


[BEFORE_START]
-    Heat the Incubators to 56°C for lysis.
-    Heat buffer PL1 at least 30min at 56°C before to start the extraction to dissolve SDS precipitant.
-    Clean all the area with DNA away.
-    Equilibrate buffer PE to 65°C.

-   Proteinase K : add 2500 µl Proteinase K buffer PB to adjust concentration to 20 mg/mL, incubate 1min     at room temperature. Don't vortex!
Do aliquots of 250µL. Store at -20°C. Don’t freeze/defreeze more than 3 times.

-    Thaw/prepare Lysozyme (20mg/ml) in TE-Buffer 1x.
   -    Wash Buffer PW2: add 100ml EtOH 96-100%

[GUIDELINES]
This protocol has been modified from the references provided by Macherey-Nagel : 

-  NucleoSpin DNA Plant II Mini kit (Macherey-Nagel, ref 740770.50) (50 preps) ;
-  NucleoSpin DNA Plant II Midi kit (Macherey-Nagel, ref 740771.20 (20 preps).

[STEPS]
1. Depending of the plankton size fraction samples you have, select :

1 -  DNA extraction using NucleoSpin Plant II MINI kits regarding [<20µ] size fractions (samples collected using Niskin bottles or pumping and filtered on polycarbonate filters, diameter 47mm) ;

2 -  DNA extraction using NucleoSpin Plant II MIDI kit regarding [>20µ] size fractions (samples collected and prefiltered using a plankton net, and filtered on polycarbonate filters, diameter 47mm).


SECTION: Lysis
3. Cut the 47mm filter into small part and put it in a 2mL sterile microtube. Clean cisors and tweezers with DNA away between each sample.
SECTION: Lysis
4. Add 25µL of proteinase K (20mg/mL). Vortex.
SECTION: Lysis
5. Add 100µl of Lysozyme (20mg/ml) to the crushed filter, vortex and incubate for 5 min at room-temperature.
SECTION: Lysis
6. Incubate 2h @ 56°C, 900rpm in the Thermomixer.
SECTION: Clarification of crude lysate
7. Transfer the lysate with the filter on a NucleoSpin Filter column (purple column).
Centrifuge 5min @ 11 000g, at room temperature.
SECTION: Clarification of crude lysate
8. Transfer the eluate on a new sterile 2mL microtube using a P1000 without touching the pellet.
SECTION: Precipitation/Purification
9. Add 550 µL of buffer PC to the lysate, mix by pipetting up and down 5 times.
Transfer 650µL of precipitate onto a green column (max 650µL).
Centrifuge 1min @ 11 000g. Discard the flow-through and replace the column on the collection tube.
SECTION: Precipitation/Purification
10. Repeat Step 9 until all liquid has been loaded.
SECTION: Precipitation/Purification
11. Add 400 µL of buffer PW1 to the membrane of the column.
Centrifuge 1min @ 11 000g. Discard the flow-through and replace the column on the collection tube.
SECTION: Precipitation/Purification
12. Add 675 µL of buffer PW2 to the membrane of the column.
Centrifuge 1min @ 11 000g. Discard the flow-through and replace the column on the collection tube.
SECTION: Precipitation/Purification
13. Add 200 µL of buffer PW2 to the membrane of the column.
Centrifuge 2min @ 11 000g. Discard the flow-through and the collection tube.
Put the column on a new sterile 1.5mL microtube correctly labeled (sample name, date, operator)
SECTION: DNA Elution
14. Add 50µL of buffer PE (preheated 65°C) onto the membrane of the column. Incubate 5 min @ 65°C in the thermomixer.
Centrifuge 1min @ 11 000g.
SECTION: DNA Elution
15. Repeat Step 13 by adding again 50µL PE or reelute the membrane with the same eluate to get more concentrated DNA.
SECTION: DNA Elution
16. Measure DNA concentration with Nanodrop or Qbit and store the DNA at -20°C (or-80°C for long-term storage).
SECTION: Lysis
3. Cut the 47mm filter into small part and put it in a 2mL sterile microtube. Clean cisors and tweezers with DNA away between each sample.
Resuspend material from the filter with buffer PL1 using a P1000.
SECTION: Lysis
2. Put 400µL of preheated buffer PL1 in a sterile microtube 2mL for each sample.
SECTION: Lysis
4. Add 250µL proteinase K 20mg/mL to each sample. Vortex.
SECTION: Lysis
5. Add 100µL lysozyme 100mg/mL. Vortex and incubate for 5 min at room-temperature.
SECTION: Lysis
6. Seal the Falcon 15mLwith parafilm and fix them on the incubator plate.
Incubate 2 h @ 56°C, under slow shaking.
SECTION: Clarification of crude lysate
7. Transfer the lysate with the filter on a NucleoSpin Filter column (purple column).
Centrifuge 10min @  4 500g, at room temperature.
SECTION: Clarification of crude lysate
8. Transfer the eluate on a new sterile Falcon 15mL using a P1000 without touching the pellet.
SECTION: Precipitation/Purification
9. Add 4,6 mL of buffer PC to the lysate, mix by vortexing 30 sec.
Transfer 3,5 mL of precipitate onto a NucleoSpin Plant Midi Column (max 4 mL) with a Collection Tube.
Centrifuge 2min @ 4 500g. Discard the flow-through and replace the column on the collection tube.
SECTION: Precipitation/Purification
10. Repeat Step 9 until all liquid has been loaded.
SECTION: Lysis
2. Put 3.4mL of preheated buffer PL1 in a Falcon 15mL for each sample.
SECTION: Precipitation/Purification
11. Add 1 mL of buffer PW1 to the membrane of the column.
Centrifuge 2min @ 4 500g. Discard the flow-through and replace the column on the collection tube.
SECTION: Precipitation/Purification
12. Add 3 mL of buffer PW2 to the membrane of the column.
Centrifuge 2min @ 4 500g. Discard the flow-through and replace the column on the collection tube.
SECTION: Precipitation/Purification
13. Add 1 mL of buffer PW2 to the membrane of the column.
Centrifuge 10min @ 4 500g. Discard the flow-through and the collection tube.
Put the column on a new sterile 15mL Falcon.
SECTION: DNA elution
14. Add 150µL of buffer PE (preheated 65°C) onto the membrane of the column. Incubate 5 min @ 65°C in the thermomixer.
Centrifuge 2min @ 4 500g.
SECTION: DNA elution
15. Add again 150µL PE (preheated 65°C) onto the membrane and centrifuge again 2min @ 4500g.
SECTION: DNA elution
16. After centrifugation, transfer volume of eluted DNA in a sterile 1.5mL microtube correctly labeled (name, date, operator) on
the both sides.
SECTION: DNA elution
17. Measure DNA concentration with Nanodrop or Qbit and store the DNA at -20°C (or-80°C for long-term storage).