Introduction
The biological samples are introduced in the PDMS device via spotting on a glass slide. The biological samples can be:
- cells, yeast (S. cerevisae) or bacterial (E. coli)
- spores, yeast (S. cerevisae) or bacterial (B. subtilis)
This protocol descibes how to prepare the cells/spores for spotting.
Procedure
S. cerevisae (Yeast GFP library strains)
- Pick a glycerol stock from the strain of choice and spread on SD-His plates. The current available strains are:
- Incubate the plate(s) at 30°C for 18-48 hr.
- Pick one colony from the strain(s) of choice and inoculate in 4-10 ml of YPD or SD-His medium.
- Incubate for 48 hr at 30°C and 130 rpm.
- Optional: Centrifuge the cells at 2400 rpm for 3 min. Resuspend the cells in 100 ul of SD-His medium.
- Place the cells in a 96-well plate for spotting.
E. coli
- Pick a glycerol stock from the strain of choice and spread on LB plates with the appropriate antibiotic (e.g. Cam). The current available strains are E. coli transformed with the following plasmids (all have Cam resistance):
- Incubate the plate(s) at 37°C overnight.
- Pick one colony from the strain(s) of choice and inoculate in 4 ml of LB medium + antibiotic (e.g. Cam).
- Incubate overnight at 37°C and 130 rpm.
- Centrifuge the cells at 3000 rpm for 5 min.
- Resuspend the cell pellet in 100 ul LB with 10% glycerol and appropriate antibiotic (e.g. Cam).
- Place the cells in a 96-well plate for spotting.
Spore preparation
S. cerevisae (Yeast GFP library strains mated with a query MATα strain)
- Place well-concentrated spores (in sporulation medium) in a 96-well plate for spotting.
- Optional: Centrifuge the spores at 2400 rpm for 3 min. Resuspend the cells in 100 ul of water.