Fluorescence in situ Hybridization Protocol (FISH for Yeast)

相关专题

 

Modified 9/96
Cell PREPARATION: (Day 1) eppendorf

Grow cells in YEPD to early to midlog phase (O.D.600 of 0.3-0.4 for haploids and 0.5-0.6 for diploids). a) Asynchronous cells: proceed to step 3.
b) Nocodazole blocked cells: add nocodazole to a final concentration of 15 ug/ml then incubate cells at 23oC for 3 hours. Go to step 3
c) Temperature sensitive mutants: transfer cells to 37℃ and incubate 3 hours. Go to step 3 Fix cells by adding 100 ul 36% formaldehyde to 1 ml of cells and incubate for 2 hours at 23℃. Transfer 1 ml fixed cells to an tube and pellet cells 20 seconds at 10 K. Resuspend cells in 1 ml distilled H2O then pellet cells 20 seconds at 10 K. Wash cells 2X more using 1 ml H2O per wash. Resuspend cells in 500 ul spheroplast buffer (At this point cells can be stored overnight at 4℃). Polylysine coat slides by adding 10 ul polylysine solution (1 mg/ml in H2O) to each well on slide. Incubate at room temperature for 10 minutes. Remove the polylysine then wash 2X with H2O and allow to air dry. Spheroplast cells by adding 10 ul beta-mercaptoethanol (1/50 cell vol), then add 5 ul (1/100 cell vol) of 3 mg/ml Zymolyase T100 (ICN or seikagiuchi zymolyase). Incubate cells for 1 hour at 23℃ in H2O bath (no shaking). Pellet cells for 5 seconds at 10 K and resuspend gently (using pipetman) in 1/2X spheroplast buffer (use a volume equal to the spheroplast volume). Add 10 ul of cells to each well. Incubate for 10 minutes at room temperature. Remove the liquid in each well using a pipetman. Slowly add 20 ul 0.5% SDS to each well and incubate for 10 minutes at room temperature. Remove SDS using pipetman (hold pipetman perpendicular in the center of the well). Allow wells to air dry (takes about 5 minutes). Place slides in a coplin jar containing 3:1 methanol:acetic acid (freshly made). Incubate for 5 minutes at room temperature. Remove slides and place them on a paper towel to and allow to air dry overnight at room temperature. (Note: I often air dry for several days until slides no longer emit an odor of acetic acid). Slides can be stored for at least 6 months at 4℃ in vacuum desiccator.

IN SITU HYBRIDIZATION: (Day 2)

RNase treatment:

Dilute RNase A stock to 100 ug/ml using 2X SSC then add 10 ul to each well. Place slides into humid chamber (prewarmed to 37℃). Incubate 1 hour at 37℃.

Dehydration: Remove slides from humid chamber and place slides in a coplin jar containing 2X SSC (room temperature). Incubate 2 minutes at room temperature (agitate slides after one minute). Wash slides 3X more (2 minutes/wash) using fresh 2X SSC. (For these washes, we prepare three coplin jars each containing 50 ml 2X SSC