McClean:Random Spore Prep
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.
- 0.01 % NP-40 (igepal) (1-2 mL/ culture)
- beta-glucoronidase (3 uL/ culture)
- sporulated diploid culture (17 uL)
- YPD or selective plates
- Spin down 250 uL of sporulated diploids at top speed for 5 min.
- Remove supernatant and resuspend in 250 uL dH20.
- Aliquot 17 uL into 2-3 labeled eppendorf tubes.
- Add 3 uL of beta-glucoronidase to each test tube.
- 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.
- 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)
Use this step to fully separate the ascii into individual haploids.
- Add 300 uL dH20 to the test tube.
- 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).
- Allow the beads to settle for 3-5 minutes.
- 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.
- Remove supernatant (being careful to avoid the beads) and pellet at top speed for 5 minutes in a clean eppendorf tube.
- Remove supernatant and resuspend in 100 uL dH20.
- Vortex for 2 min. This makes the hyprophobic haploids stick to the sides of the eppendorf.
- Wash the tubes with 1mL dH20 3X by pipetting up and down and removing water. This should remove the diploids.
- Add 1mL .01% NP-40. Sonicate for 1 min at setting 2. This may froth so let the bubbles settle before sonicating again.
- Plate 100 uL of the supernatant on the desired plate.
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
- List troubleshooting tips here.
- You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
- Anecdotal observations that might be of use to others can also be posted here.
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Adapted from: Kurt Thorn, 3/9/04
- Who has experience with this protocol?
or instead, discuss this protocol.