Maitreya Dunham
January 2005
Small batches recommended
to avoid contamination problems.
200 ml
0.5 g yeast
extract
3 g potassium
acetate
water to 200 ml
Autoclave. Once cool, add:
1.25 ml 40% glucose
20 ml 10X
amino acid stock
100 ml
40 mg adenine
40 mg uracil
40 mg tyrosine
20 mg histidine
20 mg leucine
20 mg lysine
20 mg tryptophan
20 mg methionine
20 mg arginine
100 mg phenylalanine
350 mg threonine
water to 100 ml
Filter sterilize and store
at 4C in the dark.
Streak for a fresh colony.
Grow an overnight in YPD.
Spin down ~250 µl cells
(can vary for different strains).
Decant supernatant and
resuspend pellet in 2 ml SPO++.
Transfer to a culture
tube. Also make a no cells control
to test for contamination.
Let rotate at 30C or at
room temperature, depending on the strain. I use 30C for most strains.
Check the cultures under
the microscope to see if they've sporulated.
SK1 strain will sporulate
overnight with very high efficiency.
Y55 takes maybe 2 days,
but should also be high.
S288C and CEN.PK take 3 or
more days, with low efficiency.
You'll need dissection
plates: add 25 ml YPD with a
plastic strippette to plates on a very level surface. Once solid, invert.
Let dry at room temperature for ~3 days. Bag. Best after
aging for a while.
Spin down 250 ul of
sporulated culture.
Resuspend in 250 ul
sterile water.
Mix 17 ul culture with 3
ul B-glucuronidase (can vary by strain).
Let sit 15-45 minutes
(depends on strain, culture, and ambient conditions) until digested. I generally start 2 digestions and stop
one after 20 minutes and the other after 30 and use which one is better.
Check for digestion under
the microscope.
Gently add 100 ul
water. The goal is to suspend the
cells without breaking up the tetrads.
Pick out your favorite
dissection plate. The plate should
not look wet, but when the side is deformed with your thumb, little droplets of
water should squeeze out from the surface of the agar. The droplets should be immediately
reabsorbed once the pressure is released.
Mark the center of the
plate.
Drip 20 ul down the
center.
Let absorb into the plate.
Dissect.