Prepare tetracycline LB agar plates

Making tetracycline LB agar plates

Dilute 2 uL glycerol stock in 20 mL sterile PBS (1/10000 dilution).

Pipette 100 uL of diluted sample into centre of 90 mm agar plate

Put 4-5 plating beads onto the agar. With lid on, move the plate back and forth so the beads spread the liquid evenly across the agar surface.

To remove the beads, turn the plate so the beads collect in the side of the lid. Carefully open the lid and drop the beads into a glass beaker.

Incubate the inoculated plates at 37° for ~25.5hrs.

Clean plating beads

Add sterile water to the glass beaker.

Place 70 uM cell strainer on another beaker to collect beads from liquid. Put beads back into the empty beaker. Dispose of water in waste bottle for treatment with Virkon.

Add ethanol to beads. Repeat above step. Dispose of liquid in ethanol waste bottle.

Wash beads with sterile water as above. Put beads in a bottle and send for autoclaving.

Make up 15 mg/ml tetracycline hydrochloride in sterile water, and add 1/000 dilution to LB broth (fi nal conc. 15 ug/ml)

Using the VIAFILL, dispense 50 uL of tetracycline LB per well into 384 well plates

Before using the PIXL, sterilise the instrument with UV.

Interlock door guard into slot on the inside of the instrument door Prepare growth media Pick colonies with the PIXL 4

On the Home Screen, select UV Lamp and run for 30 minutes

Set up instrument via display screen.

Home Screen -> Run Workfl ows -> Random Colony Picking -> Blank

Select Source Plate: Petri dish -> 90 mm

Capture settings -> White Light -> Select colonies

Select Target Plate: SBS -> 384

Check the Project Summary -> Start Picking

Incubate 384 well plates at 37 °C overnight.

Measure OD600 of the overnight culture using a plate reader.

Using the VIAFILL, dispense 50 uL of 30% glycerol per well into 384 well plates (final conc: 15% glycerol).

Incubate plate for 2 hours at 37 °C.

Seal using aluminium seals and store at -80 °C.