Cyanobacterial Growth, Harvest, and Genomic DNA Prep

Transfer100mLsterile 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 at100rpmbeneath 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.

• 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).

Centrifuge5000x g * Decant liquid Z8 media.

• Add50mLliquid Z8 media to tube, cap, and shake vigorously to dislodge potential bacterial contaminants from the cyanobacteria biomass.

Repeat Step 3 three times

Add a final50mLliquid Z8 media to tube.* Lightly close cap.

• Wrap with foil and place in refrigerator at4for 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)

• Centrifuge5000x g
• Remove supernatant Z8 media with a P1000 pipette and discard.

Place tubes into liquid nitrogen for 5 minutes. * Immediately transfer to-80°Cfreezer.

Thaw biomass on ice.* Transfer 500µL biomass per PowerLyzer® bead beating tube using a sterile inoculation loop .

• Place bead tube and balance in a

Equipment

Value	Label
Precellys	NAME
Homogenizer	TYPE
Bertin	BRAND
P000669-PR240-A	SKU

processing samples four times with 15 seconds of rest. 5500rpm

• 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.

• Transfer15µLPowerLyzer® elution buffer to the center of the column membrane.

• Incubate at room temperature for 3 minutes .

• Centrifuge1000x g

• Add an additional25µL elution buffer to spin column.

• Incubate at room temperature for 3 minutes.

• Centrifuge10000x g

Store genomic DNA at-20°Cuntil library prep and sequencing.