Springer Lab: Random Spore Analysis

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Spores (see Sporularion protocol)
Spores (see Sporularion protocol)
 +
10mg/mL Zymolyase-100T (zymo research, us biologicals)  
10mg/mL Zymolyase-100T (zymo research, us biologicals)  
 +
2-Mercaptoethanol (2-ME)
2-Mercaptoethanol (2-ME)
 +
1.5% (v/v) Nonidet P-40 (NP-40)
1.5% (v/v) Nonidet P-40 (NP-40)
-
Ethanol
+
 
'''Prepare the tetrads'''
'''Prepare the tetrads'''
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0.5 mL of spores resuspended in water
0.5 mL of spores resuspended in water
 +
25 microL of zymolyase
25 microL of zymolyase
 +
5 microL of 2-ME
5 microL of 2-ME
 +
'''Lyse unsporulated cells
'''Lyse unsporulated cells

Revision as of 08:28, 23 May 2012

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Adapted from Growth and Manipulation of Yeast, Douglas by A. Treco and Fred Winston. Curr. Protoc. Mol. Biol. 82:13.2.1-13.2.12. C 2008 by John Wiley & Sons, Inc

Materials

Spores (see Sporularion protocol)

10mg/mL Zymolyase-100T (zymo research, us biologicals)

2-Mercaptoethanol (2-ME)

1.5% (v/v) Nonidet P-40 (NP-40)


Prepare the tetrads

1. Microcentrifuge 0.5 ml sporulation culture for 30 seconds to 1 min until a pellet is formed. Pour off supernatant, and resuspend pellet in 5 ml water, vortex and centrifuge again. Resuspend in 0.5 mL of water.

2. Mix the following components:

0.5 mL of spores resuspended in water

25 microL of zymolyase

5 microL of 2-ME


Lyse unsporulated cells

3. Incubate overnight at 30C with gentle shaking.

Treatment of the sporulated culture with Zymolyase in a hypotonic solution results in lysis of unsporulated diploid cells. The preparation should be examined microscopically after the Zymolyase treatment to evaluate its effectiveness. A higher concentration of the enzyme or the more concentrated preparation of Zymolyase (Zymolyase-100T) can be used

4. Add 0.2 mL of 1.5% Nonidet P-40 (NP-40). Vortex tubes until most tetrads are disrupted (as observed by microscopy).

The spores should be examined after the last sonication step to ensure that no spores remain stuck together. More vortexing steps (or even sonication) at higher power settings will release the more tenacious spores. If spores remain stuck to each other, add 2 ml glass beads (Type I,Sigma) and shake 30 min at 300 rpm in an Erlenmyer flask at 30◦C. Let the beads settle and remove the supernatant containing the spores.

5. Centrifuge spores10 min at 1200×g. Aspirate or pour off supernatant and resuspend in 0.5 ml water. Vortex vigorously. Repeat.

6. Dilute the spores with water 1:1000 or get 10^3 spores/ml. Plate 100 μl on several YPD plates and incubate 1-2 days at 30C.

7. Screen spore colonies for markers of interest by replica plating

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