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These experiments are usually performed in the SK1 strain. This has a few unusual features compared to other common budding yeast lab strains:
These cells clump together (flocculate) very readily � this means that on plates they have a strange crusty consistency, while in liquid they form large particles.
To take an OD measurement, vortex culture well and pipette into cuvette, take reading immediately. Do not set up a number of cuvettes and then take readings as cells will start to settle distorting the results.
They sporulate as soon as they stop growing. Having selected diploids, freeze them and then follow the protocols below to avoid haploid contamination.
Transformations must be performed at OD<1 for good efficiency
They become petite (loose mitochondrial DNA) very easily. Before using in an experiment, grow on YPglycerol at least once.
Deletion of markers was done using hisG recombination, leaving a hisG mark.
Using different auxotrophic markers can alter sporulation rate, so always compare cells with the same set of auxotrophies.
To purify SK1 diploids:
Put a small amount of the ( strain on a YPD plate. Mix with an excess of a cells, and also put an equivalent amount of a cells in a separate patch on the plate.
Grow cells for a few hours at 30(
Make 2x 1(l spots of 5mg/ml (-factor on a YPD plate and allow to dry. Mark the sites precisely as you cannot see them once dried.
Pick a small amount cells from the centre of the patch of a cells, put into 100�l water and vortex well. Spot 1�l of this on one of the patches of �-factor. Repeat for the patch of mated cells. Incubate the plate O/N at 25(
Next day, there should be clear growth on the patch of �-factor with the mated cells, but much less on the patch of only a. If the difference is not clear, make two more spots of �-factor and spot on cells from the first round of selection as above.
Inoculate cells into 4ml YPD, grow O/N at 30(
First thing next morning, mix 500(l overnight culture with 500(l sterile 30% glycerol and freeze. Store this glycerol stock at -80(. It is wise to make multiple glycerol stocks of each strain, as you will need to use it for every experiment.
Synchronised meiosis:
Monday morning:
Patch diploid cells from glycerol stocks on a YP glycerol plate, O/N at 30(
Tuesday morning:
Patch these cells onto 4% YPD plate, leave at 30( (can leave these growing overnight and inoculate the next evening if desired).
Tuesday evening:
Inoculate cells into 5ml YPD
Wednesday evening:
Inoculate 20ml YPA with cells from the YPD overnight to OD 0.2 (for growth at 30() or 0.5 (for growth at 25(). Leave shaking overnight.
Thursday morning:
Cells should be ~OD 1.8-1.9 (4-4.5x107 cells/ml). If slightly overgrown, dilute cultures to this OD.
Spin down cells, re-suspend in the same volume of SPO media, spin, pour off and re-suspend again in the same volume of SPO media.
Shake cultures at desired sporulation temperature FAST (250rpm)
DNA replication should take place over 5-6 hours depending on temperature, rapidly followed by recombination. Monitor replication by FACS and recombination by PFGE. By the next morning most cells should form tetrads.
Notes:
Always start from the glycerol stock, and do not stop the protocol over the weekend at any point. SK1 diploids left on plates in the fridge will sporulate, which will screw up the meiosis course.
This protocol is from Adele Marston
If meiosis goes too fast, slow down the shaking speed slightly (230rpm)
Media
4% YPD plates: normal YPD with double the glucose
YP glycerol plates: YP plates with 2% v/v glycerol
Autoclave the plates minimally; they can be destroyed by over-cooking
YPA: 20g peptone
10g yeast extract
20g KOAc
Dissolve in 1000ml total volume
Filter sterilise into 2 bottles
SPO: 3g KOAc
5mg uracil
5mg histidine
25mg leucine
12.5 tryptophan
Dissolve in 1L total volume
Add 1ml filter sterilised 20% raffinose
Test pH using paper, add a few 祃 of acetic acid to pH ~7 if needed
Filter sterilise into 2 bottles
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