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 .
