TITLE: Cyanobacterial Growth, Harvest, and Genomic DNA Prep
AUTHORS: Ryan Ward, Truc Mai, Nicole Pietrasiak

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is a method for the growth of terrestrial and freshwater cyanobacteria in liquid 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.</div></div>

[STEPS]
?. [Biomass growing conditions]
Transfersterile liquid Z8 media into a sterile 250 mL polycarbonate Erlenmeyer flask (VWR® Erlenmeyer Flasks, Polycarbonate, Sterile Cat Nr. 89095-270).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 at 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.Secure flask into an orbital shaker atbeneath a fluorescent light at 35-40 μmole·m−2·s−1 and allow to grow until confluent or senescent.
100 mL
33
?. [Harvesting and preserving specimens in a biological safety hood]
?. [Harvesting and preserving specimens in a biological safety hood]
CentrifugeAddliquid Z8 media to tube, cap, and shake vigorously to dislodge potential bacterial contaminants from the cyanobacteria biomass.
Centrifuge: 5000 34, 5 min
50 mL
?. [Harvesting and preserving specimens in a biological safety hood]
?. [Harvesting and preserving specimens in a biological safety hood]
Add a finalliquid Z8 media to tube.Wrap with foil and place in refrigerator atfor 24 hours to halt photosynthesis and chromosomal replication.
50 mL
4 °C
?. [Harvesting and preserving specimens in a biological safety hood]
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) Centrifuge Remove supernatant Z8 media with a P1000 pipette and discard.Centrifuge
Centrifuge: 5000 34, 3 min
?. [Harvesting and preserving specimens in a biological safety hood]
Immediately transfer tofreezer.
-80 °C
?. [Genomic DNA Prep]
Thaw biomass on ice.Transfer   biomass per PowerLyzer® bead beating tube using a sterile inoculation loop. Place bead tube and balance in aTransfer  biomass per PowerLyzer® bead beating tube using a sterile inoculation loop. processing samples four times with 15 seconds of rest.
500 µl
Centrifuge: 5500 33, 45 s
?. [Genomic DNA Prep]
Process samples with the , following the manufacturer protocol, with a modified elution step as follows.Place spin column into sterile 1.5 mL Eppendorf tube.Transfer PowerLyzer® elution buffer to the center of the column membrane.Incubate at room temperature for 3 minutes. Centrifuge  Add an additional  elution buffer to spin column.Incubate at room temperature for 3 minutes. TransferPowerLyzer® elution buffer to the center of the column membrane.Incubate at room temperature for 3 minutes. Centrifuge  Add an additional  elution buffer to spin column.Incubate at room temperature for 3 minutes. CentrifugeAdd an additional elution buffer to spin column.Centrifuge
15 µl
Centrifuge: 1000 34, 3 min
25 µl
Centrifuge: 10000 34, 3 min
?. [Genomic DNA Prep]
Store genomic DNA atuntil library prep and sequencing.
-20 °C