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HARRY SCHERTHAN and EDGAR TRELLES-STICKEN
6. To obtain extensively spread nuclei, put the cell suspension aliquots
immediately on ice and, after spheroplasting (procedure 1.3), refer
to procedure 1.5 for further processing. To prepare undisrupted or
mildly spread nuclei, add the aliquots to I ho volume of 37% formaldehyde on ice. After 30 min perform spheroplasting (see below) and
continue as described under procedure 1.4 or 1.5.
1.3 Spheroplast preparation
1. Add 2% (v/v) of a 0.5 M DTT stock to the aliquots of the cell cultures
obtained in procedure 1.1, step 7 or procedure 1.2, step 6.
2. Add 1% (v/v) of Zymolyase lOOT Stock solution and vortex the tube
briefly.
3. Incubate for 10 - 20 min at 37°C.
4. Check degree of cell wall degradation by placing an aliquot (5-10 Jll) of
the digested suspension on to a slide.
5. Mix with an equal volume of a 2% sodium N-lauroylsarcosine.
6. Immediately cover the mixture with a cover glass and observe the sample instantly under a phase contrast microscope (l6x magnification).
7. After a few seconds the cells should be seen bursting (initially bright
cells become dark and then lyse). The cell wall digest is complete when
most of the cells (80%) rupture instantly. This is a critical indicator for
the success of FISH (see Troubleshooting).
8. Stop the digest by adding an equal volume of ice cold Solution II; sediment cells as described in procedure 1.1, step 6, and resuspend pellet in
1/ 20 volume Sol II as compared to initial aliquot volume.
9. The resulting pellet may be subjected either to preparation of threedimensionally preserved nuclei (procedure 1.4) or to detergent spreading (procedure 1.5).
1.4 Preparation of structurally preserved yeast nuclei for FISH
1. Wash spheroplasts (see procedure 1.3, step 9) two times in 1xSSC at
RT and fix once by resuspending the pellet in 70% ethanol.
HARRY SCHERTHAN and EDGAR TRELLES-STICKEN
6. To obtain extensively spread nuclei, put the cell suspension aliquots
immediately on ice and, after spheroplasting (procedure 1.3), refer
to procedure 1.5 for further processing. To prepare undisrupted or
mildly spread nuclei, add the aliquots to I ho volume of 37% formaldehyde on ice. After 30 min perform spheroplasting (see below) and
continue as described under procedure 1.4 or 1.5.
1.3 Spheroplast preparation
1. Add 2% (v/v) of a 0.5 M DTT stock to the aliquots of the cell cultures
obtained in procedure 1.1, step 7 or procedure 1.2, step 6.
2. Add 1% (v/v) of Zymolyase lOOT Stock solution and vortex the tube
briefly.
3. Incubate for 10 - 20 min at 37°C.
4. Check degree of cell wall degradation by placing an aliquot (5-10 Jll) of
the digested suspension on to a slide.
5. Mix with an equal volume of a 2% sodium N-lauroylsarcosine.
6. Immediately cover the mixture with a cover glass and observe the sample instantly under a phase contrast microscope (l6x magnification).
7. After a few seconds the cells should be seen bursting (initially bright
cells become dark and then lyse). The cell wall digest is complete when
most of the cells (80%) rupture instantly. This is a critical indicator for
the success of FISH (see Troubleshooting).
8. Stop the digest by adding an equal volume of ice cold Solution II; sediment cells as described in procedure 1.1, step 6, and resuspend pellet in
1/ 20 volume Sol II as compared to initial aliquot volume.
9. The resulting pellet may be subjected either to preparation of threedimensionally preserved nuclei (procedure 1.4) or to detergent spreading (procedure 1.5).
1.4 Preparation of structurally preserved yeast nuclei for FISH
1. Wash spheroplasts (see procedure 1.3, step 9) two times in 1xSSC at
RT and fix once by resuspending the pellet in 70% ethanol.