Yeast competent cell preparation can be: (1) used to establish yeast transformation systems; (2) used in yeast expression system construction; (3) used in yeast other molecular biology research.
| Experimental Materials | Saccharomyces cerevisiae |
|---|---|
| Reagents, kits | YPDA liquid medium distilled water glycerol dimethyl sulfoxide |
| Instruments, consumables | Petri dish centrifuge centrifuge tube refrigerator |
| Experimental procedure | First, reagents and supplies Reagent Bacteria - Yeast Extract (Fisher), bacteria - protein, fishermen, cellulose, bacteria - agar powder, deionized water, glycerol (Sigma), dimethyl sulfoxide (Sigma), polyethylene Alcohol 3350 (Sigma), lithium acetate (Sigma), salmon sperm cell DNA (Invitrogen), yeast amino acid-free nitrogen source (Sigma), 2-(N-morpholino) ethanesulfonic acid, adenine (Sigma), glucose. 2. Instrument Water bath (VWR), centrifuge (Eppendorf), 30 °C shaker and constant temperature incubator (VWR), petri dish. Second, the reagent formula 1. YPDA liquid or solid medium (1) 1% yeast extract: 10 g/L (2) 2% tryptone: 20 g/L (3) 2% glucose: 20 g/L 2. Conversion fluid (1) Polyethylene glycol 3350 (50% (w/v): 260 μl (2) 1 M lithium acetate: 36 μl (3) SsDNA (10 mg/ml): 10 μl (4) Plasmid: 3 μl (5) Total: 360 μl 3. YNB+ MA medium (100 ml) (1) YNB: 0.67 g (2) 2-(N-morpholino)ethanesulfonic acid (20 mM): 0.39 g (3) adenine: 0.01 g (4) Agar powder: 2g (5) Glucose: 2g Third, the preparation steps 1. Yeast strains were activated on plates of YPDA medium two days prior to the transformation experiment. 2. One day prior to transformation, activated yeast cells (monoclonal) were picked in YPDA liquid medium and incubated overnight at 30 ° C, 200 rpm. 3. Transfer the overnight yeast cell solution to a new YPDA liquid medium at a ratio of 1:3, incubate at 30 ° C for 4 h at 200 rpm. 4. Centrifuge the cells for 3 minutes at 3 000 g, and wash the yeast cells with 0.5 volumes of sterile water. 5. Centrifuge again at 3 000 g for 5 minutes. 6. Resuspend the yeast cells in 0.01 volumes of sterile water and transfer to a suitable centrifuge tube and centrifuge at 3 000 g for 5 minutes at 20 °C. 7. Resuspend the yeast cells with 0.01 volumes of sterile cell suspension (5 % v/v glycerol, 10% v/v dimethyl sulfoxide). 8. Dispense the resuspended yeast cells into a 1.5 ml centrifuge tube at 50 ul. 9. Place the tube in a plastic box or carton (gradual gradient freezing enhances yeast survival). 10. Place the box containing the yeast cells in a -80 °C freezer. (The cells can be stored at -80 ° C for more than one year). |
| other | First, the reference 1. Gietz RD, Schiestl RH (2007). Frozen competent yeast cells that can be transformed with high efficiency using the LiAc/SS carrier DNA/PEG method. Nat Protoc 2(1): 1-4. 2. Lu Y., Chanroj S., Zulkifli L., Johnson MA, Uozumi N., Cheung A., Sze H. (2011). Pollen tubes lacking a pair of K+ transporters fail to target ovules in Arabidopsis. Plant Cell 23 (1): 81-93. |
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