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2008-6-9 13:12
DAPI staining yeast - 1mL yeast + 0.1mL 36% formaldehyde. 1-2H/Rt
- Wash 2x 1mL H2O (Spin at 10K, 5" ok). May leave at 4oC
- To stain: Spind down and resuspend in 300 ul H2O. Add 700 ul 100% EtOH. Leave for 30 min/Rt.
- Spin down, resuspend 500 ul H2O and sonicate 5 sec.
- Spin down (10K, 5") and remove top 300 ul. Vortex.
- To see, mix 4 ul cells and 4 ul Eileen's premade DAPI + mounting media on slide. Microscope immeidately or seal with nail polish & keep dark. **Must seal if you want to take pictures.
EILEEN'S Premade DAPI/mounting media:
•50mg p-phenylenediamine (Sigma in 5mL 1X PBS; adjust to pH 9.0 (NaOH)
•add 45mL glycerol & stir to homogeneity
•add 2.25 ul 1mg/ml DAPI. Store -70oC/dark
[ ] f 45ug/ml DAPI
use 1:1 to stain cells, ie 4 ul cells + 4 ul DAPI
•Alt: Sonicate cells, mix in triton x100 (to 1% final) & seal immediately! |
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