Mar 19, 2024

Public workspaceLysis of diatom bulk samples

DOI
dx.doi.org/10.17504/protocols.io.x54v9ddkpg3e/v1
  • 1University of Duisburg-Essen, Aquatic Ecosystem Research
Till Macher
Open access
DOI: dx.doi.org/10.17504/protocols.io.x54v9ddkpg3e/v1
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
Funders Acknowledgement:
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: Amount200 mL Proteinase K working solution (10 mg/mL ReagentProteinase K7BioScienceCatalog #RP100B )
TNES buffer (Concentration50 Molarity (M) TRIS , Concentration400 Molarity (M) NaCl , Concentration20 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 ReagentZirconia Beads 1 mm diaBioSpec ProductsCatalog #11079110zx
2 mm dia zirconia beads ReagentZirconia Beads 2 mm diaBioSpec ProductsCatalog #11079124zx
2 mL screwcap tubes Reagent2 mL screwcap tubeSarstedtCatalog #72.693
50 mL Falcon tube ReagentEasy Reader Conical Polypropylene Centrifuge TubeCatalog #11512303
Sample collection (short description)
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 Amount10 mL diatom sample (e.g. scrape sample) to the Falcon tube.

Add Amount40 mL EtOH (96%) denatured to the Falcon tube.

Mix sample by inverting the Falcon tube.
Store samples under dark conditions at TemperatureRoom temperature or at Temperature4 °C until lysis.

Sample concentration
Sample concentration
7m
Centrifuge 50 mL falcon tubes containing the diatom samples Centrifigation4000 x g, 00:10:00

10m
Discard supernatant EtOH (can be poured from the falcon tube).
1m
Add Amount10 mL fresh EtOH (96%) denatured to each sample (in falcon tube). Vortex samples to elute pellets again.
1m
Sample lysis
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 Amount1 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 Centrifigation6000 x g, 00:05:00 .

5m
Discard supernatant EtOH (use 1000 µL tips).
Add Amount900 µL TNES and Amount100 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 Duration00:02:00 at Shaker2400 rpm .

2m
Incubate samples at Temperature55 °C for Duration00:20:00 at Shaker1400 rpm .

20m
Store samples at Temperature-20 °C until DNA extraction.