TITLE: DNA-extraction of Daphnia and symbionts
AUTHORS: Pascal Angst, Peter D. Fields

[DESCRIPTION]
This protocol was designed for DNA extraction of about 50 adult female Daphnia magna, it should also work for 1-150 animals but with adjusted reagent volumes. For achieving HMW DNA or maximizing yield, some modifications are indicated as substeps. Before DNA extraction, animals can be freed from microbes using antibiotics (https://www.evolution.unibas.ch/ebert/lab/daphnia_dna.htm) and should be dehydrated and snap-frozen with liquid nitrogen for archiving HMW DNA.

[STEPS]
SECTION: Tissue lysis and digest
1. Add   and 200 µL   to a 1.5 ml tube. (See abstract for how to prepare the animals.)
SECTION: Tissue lysis and digest
2. Grind animals with a clean, DNA(ase)-free plastic pestle, matching the shape of the 1.5 ml tube to maximize tissue maceration.
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3. Add 300 µL   and vortex shortly.

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4. Add 20 µL ProtK20 mg/mL  and carefully invert 25 times.

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

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6. Put sample on ice, add 20 µL RNAse A20 mg/mL  to the cooled sample, and carefully invert 25 times.

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7. Incubate at 37 °C while shaking at   for 60 min.

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8. Put sample on on ice for 1 min.

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9. Add 300 µL   and vortex for 15 s.

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10. Centrifuge for 4 min at 16000 x g. 

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

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

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13. Centrifuge for 3 min at 16000 x g.

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

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15. Centrifuge for 1 min at 16000 x g.

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

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17. Put the open tube in the vacuum centrifuge for 15 min.

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

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1.1. For HMW DNA, use snap-frozen animals and pre-cool lysis solution and tube with ice.
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2.1. For HMW DNA, use cold a pestle and only move it up and down 10 times (no twisting).
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10.1. If the pellet is not tight, put tube on ice for 5 min and   or pre-cool centrifuge at 4 °C.

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11.1. For HMW DNA, use a 70 μm mesh.
 

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12.1. For HMW DNA, carefully invert 50 times.
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12.2. For maximum yield (but not HMW), use cold isopropanol and add 2 µL glycogen. Then, put the sample in the freezer for 60 min.
SECTION: Tissue lysis and digest
14.1. For maximum yield (but not HMW), use cold ethanol.