Yeast cell nucleus preparation experiment
Yeast cell nucleus preparation experiment
The nucleus (nucleus) is the largest and most important organelle in the cell (the old junior high school textbooks believe that the nucleus is not an organelle, the university cell biology is considered an organelle, here to the university textbooks prevail), which is composed of the nuclear membrane (nuclear membrane), the nuclear skeleton (nuclear scaffold), the nucleolus (nucleolus) Several parts It is composed of several parts: nuclear membrane, nuclear scaffold and nucleolus.
Operation method
differential centrifugation
Materials and Instruments
Yeast Move 1. Inoculate the yeast in YPD medium (100 ml to 20 L) and incubate to mid-logarithmic ( OD600 ≈ 1-5) under vigorous shaking or forced aeration. Cultures were centrifuged in pre-weighed centrifuge bottles at 1,500 g for 5 min at 4°C.2. Determine the wet weight of the yeast cells, which is approximately the same as the volume of the compacted cells. The amount of bacterium will be considered as 1 volume in all subsequent steps.3. Cells were resuspended in 2-4 volumes of ice water and immediately centrifuged at 1,500 g for 5 min at 4°C. Cells were resuspended by adding 1 volume of yeast digestive enzyme buffer containing 30 mmol/l DTT and left at room temperature for 15 min. For more product details, please visit Aladdin Scientific website.
Nucleus Buffer Ficoll Buffer
Homogenizer
4. Centrifuge the cells at 1 500 g for 5 min at 4°C and resuspend them with 3 volumes of yeast digestive enzyme buffer.5. Add 2 mg (200 U) of yeast digestase-100T per ml of original compacted cells and incubate for 40 min at 30°C on a horizontal shaker at approximately 50 r/min. Determine whether the organisms are completely transformed into protoplasts by the water-osmosis breakage method, and, if protoplastification is incomplete, continue incubation until complete.6. Centrifuge the protoplasts at 1,500 g for 5 min and carefully discard the supernatant; the protoplast spheres are not as compact as the cellular precipitates.7. Gently resuspend the precipitate with 2 volumes of ice-cold yeast digestive enzyme buffer and centrifuge the protoplasts at 1,500 g for 5 min and repeat twice.
8. Resuspend cells with 0.5 volume of Nucleus Buffer.
9. Add the cell suspension dropwise to a beaker containing 15 to 25 volumes of ice-cold Ficoll's buffer; this process is performed in an ice bath or cold room with constant shaking.10. Transfer the suspension to a centrifuge tube and centrifuge at 3,000 g for 5 min at 4°C. Repeat the centrifugation several times if cellular debris is to be completely removed.11. Transfer the supernatant to a new centrifuge tube and centrifuge at 12,000 g for 20 min at 4°C. Resuspend the precipitate with 5-10 times the volume of Nucleus Buffer; centrifuge again at 12,000 g at 4°C.12. To obtain cell nucleus lysate, resuspend the nucleus precipitate with 1 volume of lysis buffer.
13. Gently resuspend the precipitate with 2 volumes of Hovenia yeast digestive enzyme buffer; do not attempt to obtain a homogeneous suspension, but simply wash the precipitate off the wall of the tube, suspend 10 to 20 times, and centrifuge at 1,500 g for 10 min to recover protoplasts.
14. Carefully resuspend the protoplast precipitate in 1 volume of lysis buffer using a glass rod.15. In a Dounce homogenizer with the tightest mortar and pestle, impact 15 to 20 times to cleave protoplasts.
16. Add half the volume of protoplast lysate to the ultracentrifuge tube, add an equal volume of extraction buffer, and seal the tube. Gently invert the tube for 15-30 min at 4°C on a rotating wheel or shaker.
17. Centrifuge at 100,000 g on a 45 Ti rotor for 90 min at 4°C. Collect the supernatant and dialyze in 100 volumes of storage buffer for 2 to 4 h. Transfer the bag to 100 volumes of fresh storage buffer and dialyze for an additional 2 to 4 hours.18. Remove a few microliters of the dialysate, dilute it 1:1,000 with water and determine the conductivity. If it is equal to or less than a similar dilution of storage buffer (usually 100-250 mmol/l NaCl), proceed to step 19, otherwise continue dialysis.19. Centrifuge the dialysate at 10,000 g for 10 min at 4°C. Collect the dialysate supernatant. The dialysate supernatant was collected and frozen in small portions in liquid nitrogen and stored at -80 °C.