| 培養基名稱: | SC-URA培養基 ，SC-U培養基，酵母SC-URA培養基、酵母SC-U培養基、酵母尿嘧啶缺陷型培養基；酵母尿嘧啶缺陷培養基、尿嘧啶缺陷篩選培養基 |
|---|---|
| 英文名稱: |
SC-Dropout Medium SC-U Medium SC Dropout Medium without uracil Yeast Synthetic Drop-out Media |
| 產品目錄號: | M300-01、M300-02、M300-08 |
| 產品規格: | 多規格 |
| 保存條件: | 密封，陰涼干燥處保存。 |
| 產品性狀: |
淺黃色粉末。 液體淺琥珀色，透明，無沉淀或有輕微沉淀（不溶性硫酸鈣）。 |
| 注意事項: | 避免攝入、呼入、皮膚接觸。配制時在通風櫥中進行，戴口罩、手套、護目鏡。 |
| 相關產品: |
酵母氮源基礎（YNB） SC-URA 氨基酸 酵母SC-Dropout氨基酸 |
酵母SC-URA培養基產品描述：
酵母SC-URA培養基又稱酵母SC-U培養基，是合成和篩選培養基，用于篩選尿嘧啶營養缺陷型的釀酒酵母，是尿嘧啶營養缺陷型培養基。
SC-URA培養基與酵母SC完全（SC Complete medium）培養基相比，缺少尿嘧啶。不能合成尿嘧啶的酵母菌株在SC-URA dropout培養基中不能生長。
本產品中不含糖類，使用者可以根據實驗目的添加葡萄糖、棉籽糖或半乳糖。
SC-URA尿嘧啶缺陷型酵母培養基用途
酵母SC-URA培養基用于篩選尿嘧啶營養缺陷型的釀酒酵母。
酵母SC-URA培養基添加半乳糖后，可作為釀酒酵母蛋白表達培養基。
酵母SC-URA培養基添加葡萄糖，可作為尿嘧啶缺陷型酵母傳代和增菌用培養基。
由于SC-U培養基成分已知，因此可以用來鑒定釀酒酵母URA3營養缺陷型。
SC-Ura酵母培養基配方與配制方法
| 成分 | g/L |
|---|---|
| 酵母基礎氮源（YNB） | 1.7 |
| 硫酸銨 | 5 |
| L-精氨酸 | 0.1 |
| L-半胱氨酸 | 0.1 |
| L-賴氨酸 | 0.1 |
| L-蘇氨酸 | 0.1 |
| L-天冬酰胺 | 0.05 |
| L-異亮氨酸 | 0.05 |
| L-苯丙氨酸 | 0.05 |
| L-脯氨酸 | 0.05 |
| L-絲氨酸 | 0.05 |
| L-酪氨酸 | 0.05 |
| L-纈氨酸 | 0.05 |
| L-甲硫氨酸 | 0.05 |
| L-色氨酸 | 0.1 |
| L-組氨酸 | 0.1 |
| L-亮氨酸 | 0.1 |
| 腺嘌呤 | 0.1 |
| YNB成分 | mg/L |
|---|---|
| 肌醇 | 2.0 |
| 煙酸(維生素B3) | 0.4 |
| 鹽酸硫胺素(維生素B1) | 0.4 |
| 硫酸銅 | 0.04 |
| 磷酸二氫鉀 | 1000 |
| 硼酸 | 0.5 |
| 鹽酸吡哆醇(維生素B6) | 0.4 |
| 泛酸鈣(維生素B5) | 0.4 |
| 對氨基苯甲酸 | 0.2 |
| 硫酸鎂 | 500 |
| 硫酸錳 | 0.4 |
| 硫酸鋅 | 0.4 |
| 氯化鐵 | 0.2 |
| 核黃素(維生素B2) | 0.2 |
| 氯化鈣 | 100/td> |
| 碘化鉀 | 0.1 |
| 鉬酸鈉 | 0.2 |
| 生物素(維生素B7、H) | 0.002 |
| 氯化鈉 | 100 |
配制方法：
1. 在分析天平上稱取SC-U培養基7.9g，加900 mL去離子水溶解。
2. 15 psi， 121°C 滅菌15 min。
3. 冷卻至50°C左右，加入100 mL的20%葡萄糖溶液（預先配制，用0.2微米濾膜過濾除菌），根據實驗需要，也可以用棉籽糖、半乳糖。
乳糖誘導的釀酒酵母蛋白表達實驗流程
GAL1 Promoter
In INVSc1, transcription from the GAL1 promoter is repressed in the presence of glucose (West et al., 1984). Removing glucose and adding galactose as a carbon source induces transcription (Giniger et al., 1985). Maintaining cells in glucose gives the most complete repression and the lowest basal transcription of the GAL1 promoter. Transferring cells from glucose- to galactose-containing medium causes the GAL1 promoter to become de-repressed and allows transcription to be induced.
Alternatively, cells may be maintained in medium containing raffinose as a carbon source. The presence of raffinose does not repress or induce transcription from the GAL1 promoter. Addition of galactose to the medium induces transcription from the GAL1 promoter even in the presence of raffinose. Induction of the GAL1 promoter by galactose is more rapid in cells maintained in raffinose when compared to those maintained in glucose.
You may choose to grow cells containing your pYES fusion vector in glucose or raffinose depending on how quickly you want to obtain your expressed protein after induction with galactose and on the toxicity of the expressed protein. For more information about expression in yeast, please refer to the Guide to Yeast Genetics and Molecular Biology (Guthrie and Fink, 1991).
誘導實驗
To induce expression of your protein of interest from the GAL1 promoter, galactose is added to the medium. For cells that have been maintained in glucose, recombinant fusion protein can be detected in as little as 4 hours after galactose induction. Recombinant fusion protein can be detected in cells that have been cultured in raffinose by 2 hours after galactose induction.
If you are assaying for expression of your recombinant fusion protein for the first time, we recommend that you perform a time course to optimize expression of your recombinant protein (e.g. 0, 4, 8, 12, 16, 24 hours after galactose induction). A standard protocol is provided below to perform a time course experiment. Other protocols are suitable.
1. Inoculate a single colony of INVSc1 containing your pYES fusion vector into 15 ml of SC-U selective medium containing 2% glucose or 2% raffinose. Grow overnight at 30°C with shaking.
2. Determine the OD600 of your overnight culture. Calculate the amount of overnight culture necessary to obtain an OD600 of 0.4 in 50 ml of induction medium (SC-U selective medium containing 2% galactose).
Example: Assume that the OD600 of an overnight culture is 3 OD600 per ml. Then, the amount of overnight culture needed to inoculate a 50 ml culture to OD600 = 0.4 is (0.4 OD/ml) (50 ml) = 6.67 ml 3 OD/ml
3. Remove the amount of overnight culture as determined in Step 2 and pellet the cells at 1500 x g for 5 minutes at room temperature. Discard the supernatant.
4. Resuspend the cells in 50 ml of induction medium. Grow at 30°C with shaking.
5. Harvest an aliquot of cells at 0, 4, 8, 12, 16, and 24 hours after addition of cells to the induction medium. For each time point, remove 5 ml of culture from the flask and determine the OD600 of each sample. You will use this information when assaying for your recombinant fusion protein.
6. Centrifuge the cells at 1500 x g for 5 minutes at +4°C.
7. Decant the supernatant. Resuspend cells in 500 µl of sterile water.
8. Transfer cells to a sterile microcentrifuge tube. Centrifuge samples for 30 seconds at top speed in the microcentrifuge.
9. Remove the supernatant.
10. Store the cell pellets at -80°C until ready to use. Proceed to the next section to prepare cell lysates to detect your recombinant protein.
4. Resuspend the cells in 50 ml of induction medium. Grow at 30°C with shaking.
5. Harvest an aliquot of cells at 0, 4, 8, 12, 16, and 24 hours after addition of cells to the induction medium. For each time point, remove 5 ml of culture from the flask and determine the OD600 of each sample. You will use this information when assaying for your recombinant fusion protein.
6. Centrifuge the cells at 1500 x g for 5 minutes at +4°C.
7. Decant the supernatant. Resuspend cells in 500 µl of sterile water.
8. Transfer cells to a sterile microcentrifuge tube. Centrifuge samples for 30 seconds at top speed in the microcentrifuge.
9. Remove the supernatant.
10. Store the cell pellets at -80°C until ready to use. Proceed to the next section to prepare cell lysates to detect your recombinant protein.
檢測目的蛋白
Detection of To detect expression of your recombinant fusion protein by western blot, you Recombinant may use the Anti-V5 or the Anti-His antibodies or an antibody to your protein of interest.
You will also need to prepare a cell lysate from your yeast transformant. A general protocol for small-scale preparation of cell lysates using acid-washed glass beads is provided below for your convenience. Other protocols are suitable. Please refer to Current Protocols in Molecular Biology, Unit 13.13 (Ausubel et al., 1994) for more information. For large-scale preparations (culture volumes over 1 liter).
Breaking buffer (50 mM sodium phosphate, pH 7.4, 1 mM EDTA, 5% glycerol, 1 mM PMSF).
Acid-washed glass beads (0.4-0.6 mm size; Sigma-Aldrich, Catalog no. G8772).
1. You may prepare cell lysates from either frozen cells or fresh cells.
Reminder: You will need to know the OD600 of your cell sample(s) before beginning (see Step 5).
2. Resuspend fresh or frozen cell pellets in 500 µl of breaking buffer. Centrifuge at 1500 x g for 5 minutes at +4°C to pellet cells.
3. Remove supernatant and resuspend the cells in a volume of breaking buffer to obtain an OD600 of 50-100. Use the OD600 determined in Step 5, previous page, to calculate the appropriate volume of breaking buffer to use.
4. Add an equal volume of acid-washed glass beads.
5. Vortex mixture for 30 seconds, followed by 30 seconds on ice. Repeat four times for a total of four minutes to lyse the cells. Cells will be lysed by shear force. You can check for the extent of lysis by checking a small aliquot under the microscope.
6. Centrifuge in a microcentrifuge for 10 minutes at maximum speed.
7. Remove supernatant and transfer to a fresh microcentrifuge tube. Assay the lysate for protein concentration using BSA as a standard.
8. Add SDS-PAGE sample buffer to a final concentration of 1X and heat the sample for 5 minutes at 70°C.
9. Load 20 µg of lysate onto an SDS-PAGE gel and electrophorese. Use the appropriate percentage of acrylamide to resolve your recombinant protein.
3. Remove supernatant and resuspend the cells in a volume of breaking buffer to obtain an OD600 of 50-100. Use the OD600 determined in Step 5, previous page, to calculate the appropriate volume of breaking buffer to use.
4. Add an equal volume of acid-washed glass beads.
5. Vortex mixture for 30 seconds, followed by 30 seconds on ice. Repeat four times for a total of four minutes to lyse the cells. Cells will be lysed by shear force. You can check for the extent of lysis by checking a small aliquot under the microscope.
6. Centrifuge in a microcentrifuge for 10 minutes at maximum speed.
7. Remove supernatant and transfer to a fresh microcentrifuge tube. Assay the lysate for protein concentration using BSA as a standard.
8. Add SDS-PAGE sample buffer to a final concentration of 1X and heat the sample for 5 minutes at 70°C.
9. Load 20 µg of lysate onto an SDS-PAGE gel and electrophorese. Use the appropriate percentage of acrylamide to resolve your recombinant protein.
放大實驗
Once you have determined the optimal induction time necessary to obtain maximal protein expression, you may increase the protein yield by scaling up the procedure. If you plan to use ProBond resin to purify your recombinant fusion protein, please see the Note below. To prepare cell lysates from culture volumes over 1 liter, we recommend that you use a bead beater to lyse the cells. Please refer to Current Protocols in Molecular Biology, Unit 13.13 (Ausubel et al., 1994) for a suitable protocol to lyse cells with a bead beater.