DNA-extraction of Daphnia and symbionts

Add Sample and 200µL to a 1.5 ml tube. (See abstract for how to prepare the animals.)

For HMW DNA, use snap-frozen animals and pre-cool lysis solution and tube with ice.

Grind animals with a clean, DNA(ase)-free plastic pestle, matching the shape of the 1.5 ml tube to maximize tissue maceration.

For HMW DNA, use cold a pestle and only move it up and down 10 times (no twisting).

Add 300µL and vortex shortly.

Add 20µL ProtK20mg/mL and carefully invert 25 times.

Incubate at 55°C while shaking at 400rpm overnight ( incubation increases yield dramatically).

Put sample On ice, add 20µL RNAse A20mg/mL to the cooled sample, and carefully invert 25 times.

Incubate at 37°C while shaking at 400rpm for 1h 0m 0s.

Put sample on On ice for 0h 1m 0s.

Add 300µL and vortex for 0h 0m 15s.

Centrifuge for 0h 4m 0s at 16000x g,0h 0m 0s.

If the pellet is not tight, put tube On ice for 0h 5m 0s and or pre-cool centrifuge at 4°C.

Pipette supernatant (800µL – 1.000µL) to a 2 ml tube. Discard tissue.

For HMW DNA, use a 70 μm mesh.

Equipment

Value	Label
pluriStrainer Mini 70 µm	NAME
Cell Strainer	TYPE
pluriSelect	BRAND
43-10070-40	SKU
https://www.pluriselect.com/	LINK

Add the same amount of isopropanol (800µL – 1.000µL) to the supernatant and carefully invert 25 times.

For HMW DNA, carefully invert 50 times.

For maximum yield (but not HMW), use cold isopropanol and add 2µL glycogen. Then, put the sample in the freezer for 1h 0m 0s.

Centrifuge for 0h 3m 0s at 16000x g,0h 0m 0s.

Discard supernatant, add 500µL 70 % ethanol, and carefully invert until the pellet dislodges.

For maximum yield (but not HMW), use cold ethanol.

Centrifuge for 0h 1m 0s at 16000x g,0h 0m 0s.

Discard supernatant.

Apply for HMW DNA and repeat step 14.-16. twice for purification.

Put the open tube in the vacuum centrifuge for 0h 15m 0s.

Add 80µL and incubate in the dark overnight. If fewer animals are being used or less DNA yield is expected, add less (20µL-50µL)