Abstract
This protocol describes the procedure to label a certain mRNA species in yeast cells with fluorescence probes and prepare slides for visualization under microscope. Probe design/synthesis and microscopy operation are not covered.
Materials and Reagents
Equipment
Procedure
Duration: DAY 2: ~3-4 h, DAY 3:~2-3 h after OD reached, DAY 4: ~1-2 h, DAY 5: ~1-2 h.
DAY 0: Prepatation of coverslips
DAY 1: Grow a 5 ml culture in appropriate media O/N on wheel at 30 °C.
DAY 2: In the morning, dilute cultures 1:10 and let them grow.
Check OD600 in the afternoon and dilute the culture acco rdingly into 50 ml of media, so that OD600 in the morning = 0.3-0.5 incubate cultures in the shaker at 30 °C O/N.
DAY 3: Fixation of yeast cells (Note: KEEP CELLS ON ICE!)
DAY 4: In situ hybridization (Note: Wear Gloves) Preparation of probes:
Hybridization:
DAY 5: Washing and mounting
Note: For the following steps, wrap coplin jars in aluminum foil to protect from light. Prepare 2x the amount of reagents (2 washes).
Notes
Recipes
References
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