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Protocol for making double deletions through mating, sporulation, tetrad dissection, and verification of spores.


This protocol will follow the example of making a tor1::LEU2 fob1::KanMX double mutant




Mating:


1) Select two strains which each have a deletion in a gene of interest in opposite mating types


a) Strain 1 genotype: tor1::LEU2, mat a, met15 LYS2


b) Strain 2 genotype: fob1::KanMX, mat alpha, MET15 lys2


2) Streak the two strains onto a patch on a YPD plate, with the two strains overlapping each other. This contact between the two strains enables them to mate with one another and form a diploid cell. Allow the cells to grow together for 4+ hours or overnight at 30°C.


3) If you are fortunate and the diploid is the only cell which is both MET+ and LYS+, you may streak the mated cells onto a –Met –Lys plate. (in this case, the diploid genotype is met15/MET15 lys2/LYS2, so only diploids will grow on these plates). Allow to grow on these plates for 6+ hours at 30°C or overnight, then re-patch onto a new –Met –Lys plate to ensure only diploids are growing. Grow overnight 30°C.


a) If you do not have the convenient Met Lys markers as above, you can select for zygotes. After growing the mating patches for 3-6 hours at 30°C, use a pick to scrape some cells into 30μl water or YPD.


              b)  Drop the 30μl of water with mated zygotes onto the top left corner of a YPD plate held vertically:


              c)  Allow to dry for >10 mins.  Then using a dissecting needle on the microscope, move cells that look like barbells or barbells with a cell growing off the middle onto different sections on the plate.  These cells will grow into diploid cells and you can follow the rest of the protocol normally.


4) Once the cells are in their diploid state, streak them onto GNA plates in small patches. Allow to grow 6+ hours or overnight at 30°C, and then take the patch and streak onto a fresh GNA plate. Grow overnight at 30°C. Remember GNA plates dry out fast, parafilm them or use immediately.




Sporulation:


1) Inoculate about 20μl worth of cells into 3ml sporulation media, or enough to make the media read at ~0.3-0.7 OD


2) Rotate cells for ~4-10 days at room temperature, or until you visually see tetrads under the microscope. Tetrads can look like this:


3) While waiting for sporulations, set aside thee plates you will need to dissect tetrads on. They need to dry outside of the metal boxes for 2-5 days before dissection. If this step is not done, the spores will stick to the needle and be impossible to get off, greatly slowing the ability to dissect tetrads.




Tetrad dissection:


1) Pipette 250μl of sporulated culture gently into an eppendorf 1.5ml tube.


2) Take a very small amount of Zymolyase and immerse into the eppendorf. A good way to do this is to take a pipette tip an dip it into the zymolyase powder until it touches the bottom of the tube, and then take it out an lightly touch the bottom of the eppendorf. This will attain optimum zymolyase conditions.


3) Flick tube gently. Incubate tube at 37°C for 20 minutes.


4) Take 30μl of the yeast suspension in the eppendorf and gently drip onto a pre-dried YPD plate as shown above. If wanted, it is ok to parafilm wrap the plate and leave at 4°C overnight and dissect the next day.


5) Separate the tetrads according to the diagram on the next page.


(separate by 5 unit increments, 10-20 tetrads per plate)


6) Allow to grow at 30°C for two days.



Tetrad analysis:
Here is the payoff portion. We want to ensure all the mutations are in the same spore and will confirm this both by replica plating and by PCR. Once again, we will use the example of making a tor1::LEU2 fob1::KanMX double mutant in BY.
Day 1
1) Replica plate your tetrad plates onto selective media. In this example, it would be –Leu and G418 plates. This will tell us which spores have our deletions; ones which grow on –Leu have a tor1 deletion and ones which grow on G418 have a fob1 deletion.
2) Replica plate onto –Met and –Lys plates as well. This tells us how the markers in each parent strain segregated; BY4742 is MET15 lys2 whereas BY4741 is met15 LYS2. Preferably if we freeze down a mat alpha strain, it should also be MET15 lys2, for example.


3) Replica plate onto two YPD plates as well. These will be used in matings.


4) Inoculate a full colony of both mat a and mat α mating testers into 300μl YPD. Spread each mating tester on a YPD plate so it will form a lawn of yeast.


5) Grow all replica plates and the lawns of mating testers overnight at 30°C.




Day 2


7) Use an Excel sheet to mark down which spores were able to grow on which selective media. A positive double deletion for this example in a mat alpha strain should grow on –Leu, G418, and –Met, but not –Lys. Save all plates at 4°C until entire procedure is complete.


8) Press both a YPD plate with your spores and a YPD plate with the lawn of a mating tester (either mat a or mat α lawn) onto a velvet. Then press a fresh YPD plate onto the velvet. Repeat for the other mating tester. It is also useful to streak a small amount of BY4741 and BY4742 in labeled patches away from the spores as positive controls for mating. Grow at 30°C overnight.




Day 3


9) Replica plate the mating tester + spore YPD plates onto B (basic) plates.




Day 4


10) Use Excel to mark which spores were able to grow on B plates. If a spore was mated with the mat α tester, the spore was mat a and should be able to grow on B plates. Use this to score spores as either mat a or mat α strains.


11) Mark which spores are both mat α and have both deletions and are MET15 lys2. Also mark which spores are met15 LYS2, have both deletions, and are mat a. Streak these onto fresh selective plates for single colonies.


12) At this point you may colony PCR spores which you think have both deletions. Be sure to include WT and single deletion controls. Remember to use internal primers for the markers in combination with gene flanking primers. You may also Smash and Grab these strains for normal PCR. These protocols are available upon request.


Day 5


13) Run agarose gels to confirm deletions.


14) For anything which is confirmed, grow them in 3-5ml liquid YPD 30°C overnight.


Day 6
15) Freeze the overnight cultures in freezer tubes. 800μl overnight and 200μl glycerol works well. Freeze at -80°C.




Recipes for above protocol:




GNA plates (6-8):

D-glucose 10g


Bacto Nutrient Broth 6g


Bacto Yeast Extract 2g


Difco Agar 4g


Water Fill to 200ml




Mix well and autoclave for 30 minutes liquid cycle. Pour into empty petri dishes when lukewarm. These plates are good for 1-2 days on your bench, if wrapped in parafilm they can last for months.




Sporulation media:



Final Concentration Stock solutions to add:


1% Potassium Acetate 10% Potassium Acetate (20g/200ml) 20 mls


0.005% Zinc Acetate 0.5% Zinc acetate (1g/200ml) 2 mls


1 x ura supplement 100 x ura supplement solution 2 mls


1 x his supplement 200 x his supplement solution 1 ml


1 x leu supplement 200 x leu supplement solution 1 ml




Fill to 200 ml with water. Filter sterilize.

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