1,816
edits
McClean: Tetrad Dissection: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
McClean: Tetrad Dissection (view source)
Revision as of 13:20, 30 September 2011
, 30 September 2011→Digestion
| Line 17: | Line 17: | ||
==Protocol== | ==Protocol== | ||
===Digestion=== | ===Digestion=== | ||
The spores are contained in a specialized cell wall called an ascus. In order to be able to separate the spores, the ascus is digested with the enzyme | The spores are contained in a specialized cell wall called an ascus. In order to be able to separate the spores, the ascus is digested with the enzyme β-glucuronidase. Ideally, the ascus is digested just enough that the spores can be separated but not so much that the spores fall apart entirely. | ||
# Spin down 250 | # Spin down 250 μL of sporulated culture. | ||
# Resuspend in 250 | # Resuspend in 250 μL sterile water. | ||
# Mix 17 | # Mix 17 μL washed culture with 3 μL β-glucuronidase (can vary by strain). Set up three tubes like this – you will stop them at different time points to check digestion. | ||
# Let sit at room temperature 20, 30, and 40 minutes. Stop the digestions as in step 5. (The ideal timing for digestion varies by strain, culture, and ambient conditions – if you are having trouble with your dissections, you may need to try less or more time. | # Let sit at room temperature 20, 30, and 40 minutes. Stop the digestions as in step 5. (The ideal timing for digestion varies by strain, culture, and ambient conditions – if you are having trouble with your dissections, you may need to try less or more time.) | ||
# Gently add 100 | # Gently add 100 μL water. The goal is to suspend the cells without breaking up the tetrads. Tap the tube gently to mix. | ||
# Transfer 5 μl of each test digestion to a slide and cover each with individual coverslips. Check for digestion under the upright microscope (see next section on appearance of tetrads). | # Transfer 5 μl of each test digestion to a slide and cover each with individual coverslips. Check for digestion under the upright microscope (see next section on appearance of tetrads). | ||
# Mark the plate at opposite edges to indicate the center of the plate (see following diagram). | # Mark the plate at opposite edges to indicate the center of the plate (see following diagram). | ||
