| Tetrad dissection四分孢子分离系统【Yale University】 | | 点击： 作者： 来源： 时间： 2007-04-11 本站论坛 |
|  | Peter Novick Lab, Department of Cell Biology Yale University School of Medicine
http://info.med.yale.edu/cellbio/Novick/Second/Protocols/Tetrad.pdf
A). Procedure:
1. Diploid has been on SPO plate (glucose plus raffinose) for 5-7 days, at
25oC. (SPO plates with sporulated yeast can be stored at 4oC for
months)
2. 100 µl tetrad juice in a test tube. Bring to room temp.
3. Take one swipe through the SPO patch with sterile toothpick. Put into
RT tetrad juice. Mix.
4. Let sit at RT for 10 minutes. Put on ice thereafter.
5. With sterile loop pick up droplet of tetrad suspension. Place droplets in
a straight line just to one side of the midline of a YDP plate. Make a
contiguous streak along that line.
6. Pick up tetrads and place each member of the tetrad on the plate such
that they are evenly spaced (about 5mm) apart.
7. Incubate 3 days at 25oC .
B). Solutions:
Tetrad Juice: store at 4oC
At final concentration: 1 M Sorbitol, 50 mM Tris, pH 7.5, 0.05 mg/ml
zymolyase.
15mls:
4 M sorbitol: 3.75 ml
1 M Tris pH 7.5 0.75 ml
zymolyase 0.75 mg
H2O 10.5 ml
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