Preparation and Mounting of Yeast Cells for Light Microscopy of

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Tony Rook
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Preparation and Mounting of Yeast Cells for Light Microscopy of

Please find the following method for:

Preparation and Mounting of Yeast Cells for Light Microscopy of Meiotic Spreads

Procedure:

1. Sporulation occurs at approximately 5 hours for SK1 derivatives and at approximately 14 hours for BR derivatives.

2. Centrifuge to pellet the cells (1,500 X g), decant the supernatant, resuspend the cells in 1 ml of 2% Acetate Solution and incubate the cells at room temperature for 10 min.

3. Add 10X Zymolyase 100T to a final concentration of 25 μg/ml. You can increase the final concentration of Zymolyase 2 to 3 fold depending on your specific conditions (this should be determined empirically).

4. Incubate the cells with Zymolyase at 30°C until 80% of the cells are sphereoplasted. A rapid way of estimating sphereoplasted is to add 5 μl of cell suspension to a standard microscope slide. Combine with 2 μl of 10% (w/v) SDS solution. Cover with a cover slip and estimate cellular lysis.

5. Centrifuge the cells at 1,500 X g and discard the supernatant.

6. To the pellet add 1 ml of ice-cold Wash Solution and mix well by inversion.

7. Centrifuge the cells and discard the supernatant

Work quickly for Steps #8, #9 and #10.

8. Resuspend the cells in 100 μl of Sulfonic Acid Solution and mix with a pipette tip.

9. Add 490 μl of Paraformaldehyde Solution.

10. Add cellular suspension to a slide and allow to stand at room temperature for 10 to 30 min.

11. Drain excess liquid from slide and add 350 μl of Paraformaldehyde solution and allow to stand at room temperature for 5 min.

12. Rinse gently with 5 ml of 0.4% (v/v) Photoflo and air dry.

13. Mount coverslips using DAPI Solution.

Solutions:

0.4% (v/v) Photoflo

DAPI Solution
70% (v/v) Glycerol
1 μg/ml DAPI in PBS Solution
2% (w/v) n-Propyl Gallate

PBS Solution
pH 7.2
2.7 mM KCl
4.3 mM Sodium Phosphate Dibasic (Na2HPO4)
137 mM NaCl
1.8 mM Sodium Phosphate Monobasic (NaH2PO4)

Paraformaldehyde Solution
Prepare just before use and let stand on ice
4% (w/v) Paraformaldehyde, pH 7.0 (CAUTION Biohazard!)
Add 10 μl of 1 M NaOH per 50 ml of solution and heat,
covered with tin foil until dissolved

Sulfonic Acid Solution
0.1 M MES
0.5 mM MgCl2
1 mM EDTA
pH 6.4 using KOH

Wash Solution
0.1 M MES
0.5 mM MgCl2
1 mM EDTA
1 M Sorbitol
pH 6.4 using KOH

Zymolyase 100T (10X)
2.5 mg/ml Zymolyase 100T

2% Acetate Solution
10 mM DTT - Add DTT just before use
0.8 M Sorbitol
2% (w/v) Potassium Acetate
pH 7.0

Polylysine Solution
Centrifuge before use to remove any insoluble material
500 μg/ml Polylysine
1 M HCl

Bioreagents and Chemicals:

Sodium Hydroxide
Magnesium Chloride
n-Propyl Gallate
Hydrochloric Acid
Polylysine
Zymolyase 100T
DTT
DAPI
EDTA
Sodium Phosphate, Dibasic
Potassium Chloride
Sodium Chloride
Glycerol
Sorbitol
Potassium Hydroxide
Potassium Acetate
Paraformaldehyde
Sodium Phosphate, Monobasic

Reference and Links:

1. Dresser ME and Giroux CN. Meiotic chromosome behavior in spread preparations of yeast. J Cell Biol. 1988 Mar;106(3):567-573.

http://www.bio.com/protocolstools/protocol.jhtml?id=p202

Arvind Singh Pundir
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Hi