Mar 19, 2024
Lysis of diatom bulk samples
- 1University of Duisburg-Essen, Aquatic Ecosystem Research
Protocol Citation: Till Macher 2024. Lysis of diatom bulk samples. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9ddkpg3e/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: November 04, 2022
Last Modified: March 19, 2024
Protocol Integer ID: 72294
Keywords: lysis of diatom bulk sample, diatom bulk sample, lysis before dna extraction, dna extraction, protocol of the leeselab, diatom, gedna project, steps of sample preparation, sample preparation, leeselab, extraction, dna, metabarcoding protocol
Funders Acknowledgements:
Umweltbundesamt
Grant ID: FKZ 3719 24 2040
Abstract
This protocol describes the steps of sample preparation and lysis before DNA extraction for the diatoms metabarcoding protocol of the LeeseLab. The protocol was established in the GeDNA project.
Guidelines
Reagents are potentially damaging to the environment. Dispose waste responsibly.
Materials
Materials required:
Below all materials needed for the protocol are listed. Vendors and part numbers are listed but interchangeable depending on the supply situation.
Chemicals:
Protk: 200 mL Proteinase K working solution (10 mg/mL Proteinase K7BioScienceCatalog #RP100B )
TNES buffer (50 Molarity (M) TRIS , 400 Molarity (M) NaCl , 20 Molarity (M) EDTA
see 'Sample preparation and lysis of homogenized malaise trap samples' (dx.doi.org/10.17504/protocols.io.dm6gpjrmjgzp/v1) for details on the stock solutions.
Labware:
1 mm dia zirconia beads Zirconia Beads 1 mm diaBioSpec ProductsCatalog #11079110zx
2 mm dia zirconia beads Zirconia Beads 2 mm diaBioSpec ProductsCatalog #11079124zx
2 mL screwcap tubes 2 mL screwcap tubeSarstedtCatalog #72.693
50 mL Falcon tube Easy Reader Conical Polypropylene Centrifuge TubeCatalog #11512303
Sample collection (short description)
Perform multi-habitat diatoms sampling (depending on the protocol).
Prepare one sterile 50 mL Falcon tube for each sample. Label the Falcon tube with EtOH resistant labels (e.g. printed labels).
Add 10 mL diatom sample (e.g. scrape sample) to the Falcon tube.
Add 40 mL EtOH (96%) denatured to the Falcon tube.
Mix sample by inverting the Falcon tube.
Store samples under dark conditions at Room temperature or at 4 °C until lysis.
Sample concentration
7m
Centrifuge 50 mL falcon tubes containing the diatom samples 4000 x g, 00:10:00
10m
Discard supernatant EtOH (can be poured from the falcon tube).
1m
Add 10 mL fresh EtOH (96%) denatured to each sample (in falcon tube). Vortex samples to elute pellets again.
1m
Sample lysis
5m
Prepare each one 2 mL twist-top tube with each one spoon of 2.0 mm (ca. 10) and 1.0 mm (ca. 30) Zirconia beads. Label tubes both on the side and the lid.
Add 1 mL sample to each twist-top tube.
Note
Either use wide-bore tips or cut off the tips. Otherwise tips tend to clock when pipetting the sample.
Centrifuge samples 6000 x g, 00:05:00 .
5m
Discard supernatant EtOH (use 1000 µL tips).
Add 900 µL TNES and 100 µL ProtK to each sample.
Note
Depending on the amount of samples this can be prepared as premix. We usually prepare TNES + Proteinase K in batches for 24 samples. Proteinase K tends to self-digest if the time for samples preparation takes too long.
Bead-beat for 00:02:00 at 2400 rpm .
2m
Incubate samples at 55 °C for 00:20:00 at 1400 rpm .
20m
Store samples at -20 °C until DNA extraction.