Yeast Nuclei Isolation
This method gives yeast nuclei which look nearly purified microscopically. Nuclei isolated in this way do not give active transcription extracts when the nuclei are salt extracted and the resulting proteins AMSO4 precipitated. The high spermidine and detergent in nuclei isolation buffer are important for isolation of intact nuclei free of cellular debris.
Cell Growth
2 liters of cells are grown in YPD (3% glucose) to A600 of 3-5. Antifoam A (two drops from a P20 pipetman) is added to media before autoclaving.
For wild type cells, 2-3 ml of YPD overnight inoculated per liter at 5:30 pm gives A600 of ~3 at 9:00 am if cells are grown at 30o. For wild-type cells grown at 25o, 10-12 ml inoculated per liter works best. Slow growing mutants need anywhere between 10-120 ml/liter inoculated depending on the strain.
Extract preparation
Harvest Cells in 1 liter bottles (4 Krpm for 10 min.). Drain excess media as well as possible and weigh cells. Expected yield is 20-35 g cells. Anything less than 18g will give poor extracts. If cells are overgrown, lyticase will work poorly in spheroplasting cells.
Resuspend cell pellets in 35 ml 50 mM Tris 7.5, 30 mM DTT. Usually this can be done by gently shaking the centrifuge bottles. Leave cells in 1 liter bottles. Incubate at 30o for 15 min.
Pellet cells (4K rpm for 8 min.) and resuspend in 20 ml YPD/S. Add 15 ml 2M sorbitol. Add 15 ml recombinant lyticase. Incubate at 30o with occasional gentle mixing.
Alternative: Instead of recombinant lyticase, can also use Zymolyase 100T from ICN. Use ~2 mg per 2 liters of cells. The amount required can vary from 2-10 mg depending on the yeast strain. If using zymolyase, add in one extra YPD/S washing step at 4 degrees.
Check progress of spheroplasting every 30 min. To check, mix 4 microliters of cells with 4 microliters 1% SDS on a glass slide. Observe the number of cell ghosts under microscope. Incubate cells until about 80% spheroplasts are obtained. This can take anywhere from 30 min. to 2 1/2 hours. If cells are spheroplasting slowly after 1 hr, an extra 5-10 ml of lyticase can be added. However, if cells were overgrown (A600 >5), the cells may never spheroplast. Spheroplasting is also somewhat strain dependent.
After spheroplasting has reached about 80%, add 100 ml YPD/S (room temp) and pellet cells (4K rpm for 10 min).
Resuspend cells in 250 ml YPD/S (room temp) and incubate at 30 degrees for 30 min. to allow cells to recover. The resuspension of spheroplasts works best if a small volume (~50 ml) of YPD/S is first added and cells are resuspended using a baking spatula. Then add the remaining YPD/S.
Pellet cells (4 K rpm for 10 min.) and resuspend in 200 ml cold YPD/S (4 degrees). Resuspend as in the previous step. Keep everything cold from this point on. Cells can be kept on ice for an hour or so if other cells are still spheroplasting.
(add an extra YPD/S wash if using zymolyase instead of lyticase)
Pellet cells (4K rpm for 10 min) and resuspend in 250 ml cold 1M sorbitol.
Pellet cells (4K rpm for 10 min) and and drain sorbitol media as well as possible (careful- sometimes the spheroplast pellet is not very tight). Resuspend in 100 ml Buffer N at 4o containing DTT and protease inhibitors. Transfer to a type A "tight" dounce. Subject nuclei to 4 strokes of the dounce.
Examine lysed yeast by DAPI staining. to 4 microliters of yeast cells, add 2 microliters of 0.5 micrograms/ml DAPI made in 1 M sorbitol. It is important that the DAPI be made in sorbitol, otherwise the DAPI solution will change the lysis state of the spheroplasts. Ideally, the nuclei should remain round and intact and be mostly free of cellular debris and unlysed cells.
Differential Centrifugation to isolate nuclei
The centrifugation steps may need to be optimized depending on the weight of yeast cells and volume of buffer N used. The objective is to spin out the large cell debris, leaving the smaller nuclei in suspension. The method below was developed using 200 ml of lysed cell suspension.
Spin dounce suspension in GSA rotor at 1,500 rpm (230 x g) for 10 min. Pour off the supernatant from the somwhat loose pellet and spin supernatant again for 10 min at 2000 rpm. The pellet should be fairly firm. Be careful to avoid any slimy stuff when decanting. Most nuclei should not pellet.
Transfer supernatant to 50 ml tubes and spin 4,000 rpm in SS34 rotor 10 min. Most nuclei should not pellet. By assaying with phase contrast and DAPI, the supernanant contains mostly nuclei with one or two small phase dark dots attached. The identity of these dots has not been determined.
The nuclei can be pelleted by centrifugation at 6,000 rpm for 12 min in SS34 rotor. Freeze on dry ice.
Buffers and solutions for nuclei isolation ( volumes given are for 6 2l extracts)
50 mM Tris, 7.5 250 ml
1.5g Tris in 250 ml H2O
pH to 7.5 with HCl
Before use, add 4.6 mg DTT/ml
YPD/S 2.5 liters at room temp. (YPD with 1M sorbitol)
25g Yeast extract
50g Bactopeptone
50g Dextrose (glucose)
455g Sorbitol
H2O to 2.5 liters total
YPD/S 1.5 liters (4 degrees)
15g Yeast extract
30g Bactopeptone
30g Dextrose
273g Sorbitol
Add H2O to 1.5 liters
1M Sorbitol (4 degrees)
273g Sorbitol
H2O to 1.5 liters
Buffer N
25 mM K2SO4
30 mM HEPES 7.6
5 mM MgSO4
1 mM EDTA
10% glycerol
0.5% NP40
7.2 mM Spermidine hexahydrate (phosphate salt)
3 mM DTT and protease inhibitors are added just prior to use
This is reportedly contaminated with proteases, so extra care is needed to wash spheroplasts. Dissolve in 50 mM Tris with 2X concentrated protease inhibitors. Incubate 10 min on ice before using. This material does not dissolve well, so keep in suspension as well as possible.
Protease inhibitors and DTT
0.1 M PMSF (100x)
16 mg/ml Ethanol
Store at -20 degrees
0.2M DTT
32 mg/ml H2O
Store frozen at -20 degrees
Benzamidine (100X)
31 mg/ml H2O.
Store frozen at -20 degrees
Leupeptin (500X)
0.15 mg/ml Ethanol.
Store at -70 degrees for less than 6 months
Pepstatin (200X)
0.28 mg/ml methanol
Store at -20 degrees.
Chymostatin (2,500X)
5mg/ml DMSO
Store frozen at -20 degrees
