Lysis of diatom bulk samples
Till Macher
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.
Steps
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 10mL (e.g. scrape sample) to the Falcon tube.
Add 40mL 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
Centrifuge 50 mL falcon tubes containing the diatom samples 4000x g
Discard supernatant EtOH (can be poured from the falcon tube).
Add 10mL to each sample (in falcon tube). Vortex samples to elute pellets again.
Sample lysis
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 1mL to each twist-top tube.
Centrifuge samples 6000x g .
Discard supernatant EtOH (use 1000 µL tips).
Add 900µL and 100µL to each sample.
Bead-beat for 0h 2m 0s at 2400rpm .
Incubate samples at 55°C for 0h 20m 0s at 1400rpm .
Store samples at -20°C until DNA extraction.
