Fixation of yeast cells for RNA-FISH
Around 10am, start a cell culture in a 50ml tube, using 10ml of CSM.
Grow for 8-10 hours in a shaker at 30 °C.
Measure OD in the evening and dilute into 250ml glass flasks, starting 45ml of CSM for overnight growth.
Aiming for OD 0.2-0.4 around 10am the next morning.
Transfer to 50ml falcon tubes.
Add 5ml of Formaldehyde, invert a few times, set at benchtop for 45min.
Spin down at 3,000rpm for 4min.
Decant and wash by pipetting with 1ml of ice-cold BufferB.
Transfer to 1.5ml tubes.
(wash 1/2).
Spin down at 3,000rpm for 4min.
(wash 1/2).
Wash once more with 1ml ice-cold BufferB.
(wash 2/2).
Spin down at 3,000rpm for 4min.
(wash 2/2).
Decant and resuspend in 1ml of BufferB.
Add 2.5ul of Zymolyase and digest at 30 °C until most of the cells turn dark when checked by phase contrast microscope.
(See more in the Guidelines).
Drop 1 μL of the cells onto a glass slide, do not cover (pressure makes it look like they digested).
Use a 20x scope with a phase contrast ring.
Check digestion progress every 30 minutes.
The cells turn black/grey from white/shiny.
Aim for 80%-90% of the cells within the view to be digested.
Do not overdigest.
Wash with 1ml ice-cold BufferB, spinning 5-6min at 2,000rpm.
(wash 1/2).
Decant and wash again with 1ml ice-cold BufferB, spinning 5-6min at 2,000rpm.
(wash 2/2).
Resuspend in 1ml of 70% Ethanol overnight at 4 °C.
