This procedure begins with the mating of two genetically different haid yeast cells. Diploids are selected by replica plating onto medium that permits growth of only the diploids. The diploids are then patched onto sation plates or asci form.
The asci are dissected with the haplos placed in a defined location on the plate. Once the resulting haplos have grown to a visible colony, the plate is replicated to selected medium to identify the genotype of the individual cells. Hi, I am Adrian Morris.
And I'm Audrey Mahe. And we're from the laboratory of Dr.Michael Satcher at the University of Concordia in Montreal. Today we'll show you a procedure for the dissection of yeast asking that are usually referred to as ra.
Use this procedure in our laboratory to instruct haplo D strains with novel genotypes and to study the effects of mixing two mutations together. So let's get started To construct a diploid strain, two haplo strains of opposite mating types are mated. The haplo strains are first struck out on appropriate media YPD in our case to produce single colonies using a sterile inoculation loop, pick a small portion of a colony from one of the two haplo strains on a new YPD plate streak the loop.
Once in a straight line across the plate, mark the direction of the streak with an arrow on the bottom of the plate. Next, pick a small portion of a colony from the other haplo strain on the same YPD plate. Streak the colony perpendicular to the first strain, crossing the first streak with the second mark, the direction of the second streak.
And note the area where the second streak crosses. The first. This area will be where the diploids are produced.
Incubate the YPD plate at 30 degrees Celsius overnight. After the overnight incubation replica plate, the YPD plate onto select medium that will selectively permit growth of the newly formed diploids. Incubate the plate overnight at 30 degrees Celsius.
Once diploids have grown on the plate, pick a single colony if possible, or a small portion of the diploid streak in streak onto the same selective medium to generate single colonies. Once the diploid strain has been obtained, pick single colonies and make small patches on sporulation plates. Incubate at 30 degrees Celsius for three to seven days to sporulate.
To check for sporulation, look at cells from each patch under a light microscope. Place cells on a microscope. Slide in five to 10 microliters of water.
Place a cover slip on top of the droplet and look for the presence of asci. If ask you or not easily found, turn the plate to the incubator. If there is effective sporulation In one of the patches, prepare a sterile 15 milliliter conical tube with 100 microliters of tetra juice.
Pick cells from this sporulation patch with a sterile three millimeter inoculation loop and place in the tetrad juice. Gently swirl the loop to allow the cells to fall off the loop and mix with the solution. Leave the cells in the tetrad juice for four to 10 minutes.
This allows for digestion of the cell wall surrounding the ASCO spores. To prepare a plate for the dissection of asci, mark the top and bottom of the inoculum region on a YPD plate. When the cells are sufficiently digested, take five microliters of digested cells and gently placed drops in a line in the inoculum region of the pre-marked YPD plate with a sterile three millimeter inoculation loop.
Very gently spread the drops one to three times in align while barely making contact with the auger. Allow the liquid to absorb into the auger. You are now ready to begin dissection of the asci.
To begin dissection of the asci, place the dissecting plate under the singer MSM system 200 micro manipulation microscope. Focus on a region of the plate where half of the field contains cells and half is empty. Slowly bring the needle into focus, such that it touches the empty region of the field.
Contact between the needle and the agar is seen as a dark black circle that slowly comes into focus. After the needle contacts the aer, pull it back a little check that no cells have been deposited in the area where the needle had touched the aer. This ensures that the needle does not contain cells from a previous use.
Go to the matrix at position A one and make a mark by piercing the auger. Again, this is to help determine the orientation of the plate. If it is taken off, the before dissection is complete.
Switch to the search mode. Locate an ascus. The four ASCUS spores from an ASCUS should still be attached to each other.
Two ASCUS spores may be slightly larger than the other two. Avoid picking asci where one ASCA spore is loose and not connected to the rest of the ASCA spores. To pick up an ascus, slowly bring the needle up to the plate.
When the shadow of the microneedle is seen, line it up with the ascus to be picked. Move the needle until the black outline of a circle is seen. Touch the tetrad with this circle and then quickly pull the needle away.
Make sure all four ask us, spores from an ascus have adhered to the needle and that no additional cells that are not part of this ascus were picked up by the microneedle. Once the ascus has been picked up by the microneedle, pull the microneedle far away from the plate and go to the matrix. For the first ascus, go to position a one.
Place the ascus down by touching the plate with the microneedle and pulling away until all four. Ask us spores come off the microneedle gently tapping of the needle arm or the platform holding the YPD plate is often useful. At this step.
Continue to locate, pick and place asci down at consecutive positions on the matrix until the desired number of ASCI has been obtained. Take note of each position where an ASCUS is relocated. When the desired number of asci has been picked, go back to each ask us and break the four cells apart by teasing with the needle, tapping the platform gently or a combination of both.
Pick up the cells leaving one behind. Transfer the remaining. Ask E to a new position in the matrix and repeat until each ask.
APO in each ascus is separated into rows of four. One dissection of asci is completed. Place the YPD plate in an incubator at 30 degrees Celsius for several days to observe the growth pattern.
Once the dissected asci have grown replica plate them to selective medium. If necessary, this is a plate that has been replica plated and both the original dissection plate and the selective plate have grown overnight. The results seen on the selective plate must be scored and the appropriate haplo cell identified and selected.
In cases where diploids cannot be identified on selective plates, one must manually select mated haplos called zygotes that are easily identified at an early stage after mating. The procedure for preparing the haplo cells and mating is identical to that already described, except that the region where the diploid should be is examined after shorter incubation times at 30 degrees Celsius. We find that four to six hours is a good starting point, but the length of time should ultimately be empirically determined.
Check for zygotes under a light microscope, zygotes will appear as dumbbell shaped cells, either with or without a bud pick cells up with a three millimeter inoculation loop and place gently in 100 microliters of sterile water. After the cells have been transferred to a pre-marked YPD plate, use a three millimeter inoculation loop to spread the drops in a line very gently, while barely making contact with the auger. Allow the liquid to absorb into the auger.
Proceed to the dissection microscope. Focus on a region of the plate where half of the field contains cells and half is empty. Slowly bring the needle into focus such that it touches the empty region of the field.
As shown previously. Check that the needle does not contain cells from a previous use. Go to the matrix at position A one and make a mark by piercing the auger.
Again to help determine the orientation of the plate. Switch to the search mode and locate a zygote. Pick up the zygote with the needle.
Place the zygotes individually on the matrix. Allow the zygotes to grow for several days. Patch the diploid cells onto sporulation plates and proceed as in part three.
The mating of two haplo strains of opposite mating types generates a diploid strain. This YPD plate shows the results of a mating between a temperature sensitive strain with a lu two ox atrophy and a wildtype strain. Digested strains are spread in the inoculum region on the left where thick growth between the two black lines is seen.
Asci that have been dissected grow evenly spaced in the matrix on the right. When this YPD plate was replicated onto an SD leucine dropout plate, we see the two to two growth for the LU two marker indicating that a valid ASCO spore was dissected. The original YPD plate was also replicated onto another YPD plate and incubated at a higher temperature of 37 degrees Celsius, where the two to two growth further validates that an ASCO spore was dissected.
We've just shown you how to make two haid cells and select for diploids. Based on two different methods, one uses tropic markers and the other is based on cell morphology. In using this procedure, it so, so important to remember to select the newly constructed haid cells from a tetra dissection, which the atrophic markers are represented as predicted.
It's also important to remember to not over digest or under digest. The tetra as asci failed to stay together in tetrads, which have been over digested and are difficult to break apart in tetrads that have been under digested. So that's it.
Good luck with your experiments.
