Make 500mL YE with 10g agar and autoclave. Add 10mL adenine 50x and
5mL uracil 100x when solution
cooled down to 50-60 degrees. In the meantime, prepare stock for antibiotics at 1000x--G418 (60ug/mL) and
cycloheximide (10ug/mL) can be dissolved in sterile distilled water while anisomycin (20ug/mL) requires 50%
sterile distilled water and 50% absolute ethanol for it to dissolve. Using 50mL centrifuge tubes, mix 10uL in
40mL YES and pour (1 plate requiring 20mL).
Also, the cells must be grown in 2mL YES overnight by shaking for the following day.
When the plates are ready the next day, serial 5-fold dilutions are
spotted onto the plates in this order (32,000 cells,
6,250 cells, 1,250 cells, 250 cells, 50 cells, and 10 cells) in order to check antibiotic sensitivity. 5uL is sufficient for
each spot and since 32,000 cells/5uL, 6,400,000 cells/1mL. Use the hemacytometer to count the cells and then
multiply that number by 10^4 and then again by 200. Use this number to figure out the ratio at which the original
cells must be diluted. Using this as the initial 32,000 cells, dilute accordingly and then spot them onto the plates.