Apr 23, 2024
DNA-extraction of Daphnia and symbionts
- Pascal Angst1,
- Peter D. Fields1
- 1University of Basel
Protocol Citation: Pascal Angst, Peter D. Fields 2024. DNA-extraction of Daphnia and symbionts. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl82n96l2w/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 27, 2024
Last Modified: April 23, 2024
Protocol Integer ID: 97452
Keywords: DNA Extraction, Daphnia, Crustacean
Abstract
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.
Attachments
Protocol materials
Cell Lysis SolutionQiagenCatalog #1045696
In 2 steps
Protein Precipitation SolutionQiagenCatalog #1045697
Step 9
DNA Hydration SolutionQiagenCatalog #1045698
Step 18
SRE KitPacBioCatalog #102-208-300
Step 16.1
Tissue lysis and digest
Tissue lysis and digest
19h 31m 5s
Add animals and 200 µL Cell Lysis SolutionQiagenCatalog #1045696 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.
10s
For HMW DNA, use cold a pestle and only move it up and down 10 times (no twisting).
Add 300 µL Cell Lysis SolutionQiagenCatalog #1045696 and vortex shortly.
Add 20 µL ProtK20 mg/mL and carefully invert 25 times.
30s
Incubate at 55 °C while shaking at 400 rpm overnight (Overnight incubation increases yield dramatically).
17h
Put sample On ice , add 20 µL RNAse A20 mg/mL to the cooled sample, and carefully invert 25 times.
30s
Incubate at 37 °C while shaking at 400 rpm for 01:00:00 .
1h
Put sample on On ice for 00:01:00 .
1m
Add 300 µL Protein Precipitation SolutionQiagenCatalog #1045697 and vortex for 00:00:15 .
15s
Centrifuge for 00:04:00 at 16000 x g .
4m
If the pellet is not tight, put tube On ice for 00:05:00 and go to step #10 or pre-cool centrifuge at 4 °C .
5m
Pipette supernatant (800 µL – 1.000 µL ) to a 2 ml tube. Discard tissue.
For HMW DNA, use a 70 μm mesh.
Equipment
pluriStrainer Mini 70 µm
NAME
Cell Strainer
TYPE
pluriSelect
BRAND
43-10070-40
SKU
Add the same amount of isopropanol (800 µL – 1.000 µL ) to the supernatant and carefully invert 25 times.
30s
For HMW DNA, carefully invert 50 times.
For maximum yield (but not HMW), use cold isopropanol and add 2 µL glycogen. Then, put the sample in the freezer for 01:00:00 .
1h
Centrifuge for 00:03:00 at 16000 x g .
3m
Discard supernatant, add 500 µL 70 % ethanol, and carefully invert until the pellet dislodges.
10s
For maximum yield (but not HMW), use cold ethanol.
Centrifuge for 00:01:00 at 16000 x g .
1m
Discard supernatant.
Apply SRE KitPacBioCatalog #102-208-300 for HMW DNA and repeat step 14.-16. twice for purification.
Put the open tube in the vacuum centrifuge for 00:15:00 .
15m
Add 80 µL DNA Hydration SolutionQiagenCatalog #1045698 and incubate in the dark overnight. If fewer animals are being used or less DNA yield is expected, add less (20 µL -50 µL )