You can see all details in
"Genetic and Molecular Analysis of Mitotic Recombination in Saccharomyces cerevisiae"
Protocol DNA Recombination
Volume 745 of the series Methods in Molecular Biology pp 151-172
You can see the chaper: The Sister Chromatid Exchange (SCE) Assay
Dawn M. Stults , Michael W. Killen , and Andrew J. Pierce.
Phouthone Keohavong and Stephen G. Grant (eds.), Molecular Toxicology Protocols, Methods in Molecular Biology,
vol. 1105, DOI 10.1007/978-1-62703-739-6_32, © Springer Science+Business Media New York, 2014.
1. Grow cells in 10 µM BrdU for two cell divisions (40 hrs for 293 cells). Cover plates with aluminum foil (BrdU is light sensitive).
2. Add Colcemid to a final concentration of: 150 ng/mL (13,333X of 2 mg/mL stock, ie 0.75 µL per 10 mL) for the final 30 min to enrich mitotic cells.
3. Transfer medium (floating cells) and trypsinized cells to a new tube. Spin cells at 180 xg for 10 min.
4. Aspirate supernatant leaving 0.2 mL of media and resuspend cells by gently tapping bottom of tube.
5. Add 6 mL of prewarmed 75 mM KCl dropwise for first 1mL while gently vortexing. Incubate 16 min at 37ºC (incubator).
6. Add four drops of fixative, gently invert, and spin down cells at 180 xg for 10 min.
7. Aspirate supernatant leaving 0.2 mL of hypotonic solution. Resuspend pellet by gently tapping and add 5 mL of freshly prepared fixative (3:1 solution of methanol:glacial acetic acid), dropwise for the first 1 mL while gently vortexing. Incubate 20 min at 4ºC. Spin down cells at 180 xg for 10 min.
8. Repeat step 6 two or three times until the cell pellet is colorless.
9. Resuspend the pellet in a small volume of fixative (
- Badges
- Science topic
- Similar topics
- Mathematics