Appendix 2. DNA extraction and amplification protocols

BioStandard DNA extraction procedure using DNeasy Blood & tissue kit (Qiagen, product nr. 69506).

1. Place filters used to filter water samples into a 1.5 ml microcentrifuge tube. Add 180 μl of buffer ATL to the sample. Ensure that the filters are completely covered by the buffer solution. Add glass bead and vortex for one minute. 
2. Add 20 μl proteinase K to the tube. Mix thoroughly by vortexing and incubate at 56°C on a hotplate until the sample is completely lysed. Vortex occasionally during incubation to disperse the sample. Lysis is completed in 3 hrs. 
3. After sample lysis add 4 μl RNase A (at a concentration of 100 mg/ml) to ensure that only RNA-free genomic DNA will be acquired. Mix by vortexing and incubate for 2 min at room temperature (15–25 °C) before continuing. 
4. Add 400 μl of buffer AL + ethanol. Buffer AL + ethanol and sample should be mixed immediately and thoroughly by vortexing or pipetting to yield a homogeneous solution. 
5. Pipet the mixture from the previous step (including any precipitate) into the DNeasy Mini spin column placed in a 2 ml collection tube. Centrifuge at 13 2000 rpm for 1 min. Discard flow-through and collection tube. 
6. Place the DNeasy Mini spin column in a new 2 ml collection tube, add 500 µl Buffer AW1, and centrifuge for 1 min at 132 000 rpm . Discard flow-through and collection tube. 
7. Place the DNeasy Mini spin column in a new 2 ml collection tube, add 500 µl Buffer AW2, and centrifuge for 3 min at 20,000 x g (14,000 rpm) to dry the DNeasy membrane. Discard flow-through and collection tube.
   Following the centrifugation step, remove the DNeasy Mini spin column carefully so that the column does not come into contact with the flow-through since this will result in a carryover of ethanol. If carryover of ethanol occurs, empty the collection tube, then reuse it in another centrifugation for 1 min at 20,000 x g (14,000rpm). 
8. Place the DNeasy Mini spin column in a clean 1.5 ml microcentrifuge tube, and pipet 100 µl Buffer AE directly onto the DNeasy membrane. Incubate at room temperature for 1 min, and then centrifuge for 1 min at ≥ 6,000 x g (8,000 rpm) to elute.  
   Repeat elution once as described in this step as it leads to increased overall DNA yield. 

DNA amplification

1. Calculate volumes of PCR Master Mix based on the amount of extracted DNA samples. Add a little reserve (number of samples + three (depending on the number of samples)).
2. Mixing the PCR Master Mix into 1.5 mL Eppendorf tubes. Start with the highest volume ingredient (water) and continue with lower volumes. Lastly, add the HotFire polymerase. Polymerase should remain on ice as long as possible.
3. Add 23 μL of Master mix into each of the 200 μL tubes representing different samples, subsamples, and replicates.
4. In the last step add 2 µL of your extracted DNA to the PCR mixture. The total reaction volume is now 25 μL.
5. For negative control, one PCR sample without DNA must be tested for the components' purity. In the negative control tube, add 2 µL of dH2O instead of 2 µL of DNA.

6. Centrifuge and place PCR tubes into PCR machine and start suitable PCR program (See the table below).

• 95 ˚C for 15 minutes – polymerase activation – high temperature activates DNA polymerase, which is not enzymatically active at lower temperatures. This stage also prevents extension of non-specifically bound primers and cleaves at a low-level temperature (during the preparation of the PCR reaction mixture) of the primer's dimers.
• 95 ˚C for 20 seconds – DNA denaturation – this step breaks down the weak hydrogen bonds that hold DNA strands together as a double helix. The result is single-stranded DNA to which primers can bind.
• 58 ˚C for 30 seconds – Primer's binding (annealing) – temperature lowering from 95 ˚C to 58 ˚C allows the primers to bind to the complementary sequence on the template DNA.
• 72 ˚C for 40 seconds – Elongation – 72 ˚C is for DNA polymerase to function at optimum temperature. DNA polymerase extends primers by adding a primer to the ends of free deoxynucleotide triphosphates (dNTPs) in solution and using template DNAs as an example.
• Repeat steps 2 to 4 29 times.
• Finally, another five minutes at 72 ˚C to extend the incomplete PCR products.