Feb 03, 2022

Public workspaceCyanobacterial Growth, Harvest, and Genomic DNA Prep V.2

DOI
dx.doi.org/10.17504/protocols.io.b4k2quye
  • 1Plant and Environmental Sciences Department, New Mexico State University, Las Cruces, New Mexico;
  • 2Laboratory of Genetics, University of Wisconsin‐Madison, Madison, Wisconsin;
  • 3New Mexico State University
  • Dryland Microbes Lab
Ryan D Ward
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.b4k2quye
External link: https://doi.org/10.1128/MRA.00258-21
Protocol Citation: Ryan D Ward, Truc Mai, Nicole Pietrasiak 2022. Cyanobacterial Growth, Harvest, and Genomic DNA Prep. protocols.io https://dx.doi.org/10.17504/protocols.io.b4k2quyeVersion created by Ryan D Ward
Manuscript citation:
Ward RD, Stajich JE, Johansen JR, Huntemann M, Clum A, Foster B, Foster B, Roux S, Palaniappan K, Varghese N, Mukherjee S, Reddy TBK, Daum C, Copeland A, Chen IA, Ivanova NN, Kyrpides NC, Shapiro N, Eloe-Fadrosh EA, Pietrasiak N, Metagenome Sequencing to Explore Phylogenomics of Terrestrial Cyanobacteria. Microbiology Resource Announcements 10(22). doi: 10.1128/MRA.00258-21
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: February 02, 2022
Last Modified: February 03, 2022
Protocol Integer ID: 57722
Keywords: Cyanobacteria, DNA extraction, MiSeq, Metagenomes
Abstract
This protocol is a method for the growth of terrestrial and freshwater cyanobacteria in liquid Z8 medium and the subsequent harvesting and genomic DNA extraction. Life history traits of these microorganisms such as firm cell walls, exopolysaccharide secretions, and variability in growth rates present challenges to studying their genotype. Our approach establishes a generalizable protocol to grow diverse cyanobacteria under the same conditions and a robust DNA extraction technique that produces high-quality low to medium molecule size DNA for Illumina genome sequencing.
Protocol materials
ReagentSterile 250mL Polycarbonate Erlenmeyer FlaskVWR International (Avantor)Catalog #89095-270
ReagentQiagen DNeasy® PowerLyzer® Microbial KitQiagenCatalog #12255-50
Biomass growing conditions
Biomass growing conditions
2w
2w
  • TransferAmount100 mL sterile liquid Z8 media into aReagentSterile 250mL Polycarbonate Erlenmeyer FlaskVWR InternationalCatalog #89095-270 .
CITATION
Carmichael, W. W. (1986). Isolation, culture, and toxicity testing of toxic freshwater cyanobacteria (blue-green algae). In Shilov, V. [Ed.] Fundamental research in homogenous catalysis. Volume 3. Gordon & Breach, New York, pp. 1249–62.
  • Label the flask with the strain ID.
  • Inoculate with cyanobacterial specimen.
  • Set vented cap with a 0.22µm pore-size PTFE membrane to the "open" position to allow for gas exchange.
  • Secure flask into an orbital shaker atShaker100 rpm beneath a fluorescent light at 35-40 μmole·m−2·s−1 and allow to grow until confluent or senescent.
  • Growth period may vary from 2 - 8 weeks depending on growth rate of cyanobacteria species.


2w
Harvesting and preserving specimens in a biological safety hood
Harvesting and preserving specimens in a biological safety hood
1d
1d

  • Gently pour cyanobacterial biomass of a particular species into a labelled sterile 50 mL conical tube (Falcon® Centrifuge Tubes, Polypropylene, Sterile, Corning VWR Cat Nr. 21008-936).
  • CentrifugeCentrifigation5000 x g, 00:05:00
  • Decant liquid Z8 media.
  • AddAmount50 mL liquid Z8 media to tube, cap, and shake vigorously to dislodge potential bacterial contaminants from the cyanobacteria biomass.
  • Repeat Step 3 three times
  • Add a finalAmount50 mL liquid Z8 media to tube.
  • Lightly close cap.
  • Wrap with foil and place in refrigerator atTemperature4 °C for 24 hours to halt photosynthesis and chromosomal replication.

  • Carefully decant remaining liquid Z8 media without centrifugation.
  • Retrieve sample from tubes with a sterile inoculation loop (Globe Scientific Sterile Rigid Inoculating Loops ThermoFisher Cat Nr. 22-170-204) into sterile 1.5 mL Eppendorf tubes (Fisherbrand™ Locking-Lid Microcentrifuge Tubes with Polypropylene Snap-Cap Cat Nr.: 02-681-284)
  • CentrifugeCentrifigation5000 x g, 00:03:00
  • Remove supernatant Z8 media with a P1000 pipette and discard.
  • Place tubes into liquid nitrogen for 5 minutes.
  • Immediately transfer toTemperature-80 °C freezer.
Genomic DNA Prep
Genomic DNA Prep
1h
1h
  • Thaw biomass on ice.
  • Transfer ca. Amount500 µL biomass per PowerLyzer® bead beating tube using a sterile inoculation loop.
  • Place bead tube and balance in the homogenizer.
Equipment
Precellys
NAME
Homogenizer
TYPE
Bertin
BRAND
P000669-PR240-A
SKU
https://www.bertin-instruments.com/product/sample-preparation-homogenizers/precellys24-tissue-homogenizer/
LINK
Process samples during four stages, then allow the homogenizer to cool down before turning off.
Apply the following setup:

Centrifigation5500 rpm, 00:00:45 , pauseDuration00:00:05
Centrifigation5500 rpm, 00:00:45 , cool downDuration00:05:00
Centrifigation5500 rpm, 00:00:45 , pauseDuration00:00:05
Centrifigation5500 rpm, 00:00:45 , cool downDuration00:05:00

13m 10s

  • Process samples with the ReagentQiagen DNeasy® PowerLyzer® Microbial KitVWR InternationalCatalog #12255-50 , following the manufacturer protocol, with a modified elution step as follows.


  • Place spin column into sterile 1.5 mL Eppendorf tube.


  • TransferAmount15 µL PowerLyzer® elution buffer to the center of the column membrane.
  • Incubate at room temperature for 3 minutes.
  • CentrifugeCentrifigation1000 x g, 00:03:00

  • Add an additionalAmount25 µL elution buffer to spin column.
  • Incubate at room temperature for 3 minutes.
  • CentrifugeCentrifigation10000 x g, 00:03:00



Store genomic DNA atTemperature-20 °C until library prep and sequencing.

Citations
Step 1
Carmichael, W. W.. Isolation, culture, and toxicity testing of toxic freshwater cyanobacteria (blue-green algae)