Yeast protoplast preparation experiment
Yeast protoplast preparation experiment
Protoplasts are formed by the differentiation of protoplasm, specifically the cell membrane and intramembranous cytoplasm and other life-active organelles. In plants and animals, these are the nucleus, mitochondria, and Golgi apparatus, while in bacteria, they are the ribosome and the nucleus.
Operation method
basic program
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.
YPD Yeast Digestion Buffer Sorbitol Extraction Buffer Storage Buffer Liquid Nitrogen
Shaker Water Bath Rotor Centrifuge Incubator
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. Gently resuspend the precipitate with 2-body Hovenia yeast digestase buffer; do not attempt to obtain a homogeneous suspension, but simply wash the precipitate off the wall of the tube, suspend 10-20 times, and centrifuge at 1,500 g for 10 min to recover the protoplasts.
9. Carefully resuspend the protoplast precipitate in 1 volume of lysis buffer using a glass rod.10. In a Dounce homogenizer with the tightest mortar and pestle, impact 15 to 20 times to cleave protoplasts.
11. 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.
12. 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 2-4 h. Transfer the bag to 100 volumes of fresh storage buffer and dialyze for an additional 2-4 hours.13. 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 14, otherwise continue dialysis.14. 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.