TITLE: Metagenomic library plates
AUTHORS: Bonnie Evans

[DESCRIPTION]
Arraying Canadian MetaMicroBiome Library (Neufeld et al. 2011) clones into multi-well plates for screening. 

[BEFORE_START]
Make up 30% glycerol in sterile water and autoclave.

[STEPS]
SECTION: Plate pooled library sample
1. Prepare tetracycline LB agar plates
 

SECTION: Plate pooled library sample
2. Dilute 2 uL glycerol stock in 20 mL sterile PBS (1/10000 dilution). 

 

SECTION: Plate pooled library sample
3. Pipette 100 uL of diluted sample into centre of 90 mm agar plate
SECTION: Plate pooled library sample
4. 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. 
SECTION: Plate pooled library sample
7.2. 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.
SECTION: Plate pooled library sample
7.3. Add ethanol to beads. Repeat above step. Dispose of liquid in ethanol waste bottle.
SECTION: Plate pooled library sample
7.4. Wash beads with sterile water as above. Put beads in a bottle and send for autoclaving.
SECTION: Pick colonies with PIXL
10.1. Interlock door guard into slot on the inside of the instrument door Prepare growth media Pick colonies with the PIXL 4
SECTION: Pick colonies with PIXL
10.2. On the Home Screen, select UV Lamp and run for 30 minutes
SECTION: Pick colonies with PIXL
11.1. Home Screen -> Run Workfl ows -> Random Colony Picking -> Blank
SECTION: Pick colonies with PIXL
11.2. Select Source Plate: Petri dish -> 90 mm
SECTION: Pick colonies with PIXL
11.3. Capture settings -> White Light -> Select colonies

 

SECTION: Pick colonies with PIXL
11.4. Select Target Plate: SBS -> 384
SECTION: Pick colonies with PIXL
11.5. Check the Project Summary -> Start Picking

 

SECTION: Plate pooled library sample
5. 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. 
SECTION: Plate pooled library sample
6. Incubate the inoculated plates at 37° for ~25.5hrs. 

 

SECTION: Plate pooled library sample
7.1. Add sterile water to the glass beaker.
SECTION: Pick colonies with PIXL
10. Before using the PIXL, sterilise the instrument with UV.
 

SECTION: Prepare growth media
8. Make up 15 mg/ml tetracycline hydrochloride in sterile water, and add 1/000 dilution to LB broth (fi nal conc. 15 ug/ml)
SECTION: Prepare growth media
9. Using the VIAFILL, dispense 50 uL of tetracycline LB per well into 384 well plates
SECTION: Pick colonies with PIXL
11. Set up instrument via display screen.
SECTION: Make glycerol stock plates
12. Incubate 384 well plates at 37 °C overnight.
SECTION: Make glycerol stock plates
13. Measure OD600 of the overnight culture using a plate reader.
SECTION: Make glycerol stock plates
14. Using the VIAFILL, dispense 50 uL of 30% glycerol per well into 384 well plates (final conc: 15% glycerol).
SECTION: Make glycerol stock plates
15. Incubate plate for 2 hours at 37 °C.
SECTION: Make glycerol stock plates
16. Seal using aluminium seals and store at -80 °C.
SECTION: Plate pooled library sample
7. Clean plating beads