McClean:Random Spore Prep

Overview

A way to enrich for haploid cells from a mix of haploids and diploids. This is an alternative to tetrad dissection, which can save time when a large number of strains are involved.

Materials

1. 0.01 % NP-40 (igepal) (1-2 mL/ culture)
2. dH20
3. beta-glucoronidase (3 uL/ culture)
4. sporulated diploid culture (17 uL)
5. YPD or selective plates

Spore Digestion

1. Spin down 250 uL of sporulated diploids at top speed for 5 min.
2. Remove supernatant and resuspend in 250 uL dH20.
3. Aliquot 17 uL into 2-3 labeled eppendorf tubes.
4. Add 3 uL of beta-glucoronidase to each test tube.
5. Allow reactions to proceed from 30-50 minutes, then quench the reaction by adding 100 uL dH20. It is useful to run 2 or 3 of these in parallel and check when the ascii have dissolved in 10-15 minute increments i.e 30, 40 and 50 minutes.
6. Check for digestion under a light microscope. Make sure the ascii have begun to open. 35 minutes was optimal for newly sporulated cells. (<7 days at 30 degrees)

Spore Separation

Use this step to fully separate the ascii into individual haploids.

1. Add 300 uL dH20 to the test tube.
2. Add ~100 uL of small glass beads (0.5 mm)to the tube and vortex for 2 min at top speed (this breaks up the ascii).
3. Allow the beads to settle for 3-5 minutes.
4. Remove 5-10 uL of the supernatant and inspect under light microscope for the presence of ascii. There should be few to none at this point. If there are many mostly intact ascii vortex again.

Spore Enrichment

1. Remove supernatant (being careful to avoid the beads) and pellet at top speed for 5 minutes in a clean eppendorf tube.
2. Remove supernatant and resuspend in 100 uL dH20.
3. Vortex for 2 min. This makes the hyprophobic haploids stick to the sides of the eppendorf.
4. Wash the tubes with 1mL dH20 3X by pipetting up and down and removing water. This should remove the diploids.
5. Add 1mL .01% NP-40. Sonicate for 1 min at setting 2. This may froth so let the bubbles settle before sonicating again.
6. Plate 100 uL of the supernatant on the desired plate.

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

1. List troubleshooting tips here.
2. You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
3. Anecdotal observations that might be of use to others can also be posted here.

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References

Adapted from: Kurt Thorn, 3/9/04

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