Mar 21, 2024

Public workspaceMetagenomic library plates

DOI
dx.doi.org/10.17504/protocols.io.5jyl8p328g2w/v1
  • 1MRC Laboratory of Medical Sciences;
  • 2Imperial College London
Bonnie Evans
Open access
DOI: dx.doi.org/10.17504/protocols.io.5jyl8p328g2w/v1
Protocol Citation: Bonnie Evans 2024. Metagenomic library plates. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl8p328g2w/v1
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: March 08, 2024
Last Modified: March 21, 2024
Protocol Integer ID: 96376
Abstract
Arraying Canadian MetaMicroBiome Library (Neufeld et al. 2011) clones into multi-well plates for screening.
Attachments
PIXL_user_guide_v2.9[Cleaver1].pdf
PIXL_user_guide_v2.9...
14.4MB
Materials
Tetracycline hydrochloride (Sigma-Aldrich, T3383-25G)
ColiRollers Plating Beads (Novagen, 71013-3)
384 Well Clear Flat Bottom Polystyrene Not Treated Microplate with Lid Sterile (Corning, 3680)
Before start
Make up 30% glycerol in sterile water and autoclave.
Plate pooled library sample
Plate pooled library sample
Prepare tetracycline LB agar plates
Protocol
Making tetracycline LB agar plates
NAME
Making tetracycline LB agar plates
CREATED BY
Bonnie Evans

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

Note
Keep sample on ice and return to -80 °C before proceeding with next steps.

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.

Expected result
Individual white colonies. Large enough to be detected by the PIXL but not touching neighbouring colonies.

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.
Incubation
Wash beads with sterile water as above. Put beads in a bottle and send for autoclaving.
Prepare growth media
Prepare growth media
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
Pick colonies with PIXL
Pick colonies with PIXL
Before using the PIXL, sterilise the instrument with UV.
Note
See attachment for PIXL colony picker (Sanger) user guide,

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

Note
PIXL will now analyse the image to detect colonies. You can manually select or deselect colonies, or change the algorithm to optimise detection.

Select Target Plate: SBS -> 384
Check the Project Summary -> Start Picking

Note
Check the pick up line is touching the colony and then the LB media in the well to ensure proper inoculation.

Make glycerol stock plates
Make glycerol stock plates
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.