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Mating/Sporulation to Get Difficult Strains
- Grow patches on YPD overnight.
- Pick equal amounts of the two strains into YPD Eppendorf and
rock gently or mix on YPD plate.
- Mating takes 5 h to overnight, therefore can do it overnight
but best to check for zygotes at 5-8 h to ensure there is mating
going on (zygotes often very diluted after overnight, so more
difficult to see).
- Plate various dilutions on YPD to get separated colonies —
grow till quite large.
- Pick (20-40?) colonies and patch quite densely on sporulation
plates (also spread used loop on YPD to keep strain for possible
future use). N.B. Dips grow faster, so big colonies more likely
dip but sometimes one MAT type gives bigger colonies so select
a big-biased mixture.
- Sporulation takes 1-5 d — check under microscope for tetrads.
- Release spores from one whole positive patch (no need for high
percentage of tetrads — separate protocol). N.B. If patch
too small make bigger one of positives from YPD plate or mix several
positive patches.
- Plate spores in various dilutions to get separated colonies.
- Pick random colonies (many, e.g. 100+ for rare combinations)
into 100 µl dH2O with yellow box unplugged tip
and leave tip in Eppendorf.
- Mix and drop 8 µl using same tip onto various selection
plates including YPD for keeping strain.
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– e.g. Several YPD plates for ts test
– e.g. YPgal for galHO + hmlΔ
+ hmrΔ
(+ves are non-survivors) |
Or, instead, just patch colonies on YPD plates and replica plate
onto selection plates after growth (but this loses a day and ts
test and survival not so clear).
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