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[求助]
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求助各位大神有沒有知道乳酸乳球菌MG1363感受態(tài)的具體做法及原理，和電轉(zhuǎn)方法及原理！謝謝！ @youlinglyw @wizardfan @biostar2009 發(fā)自小木蟲Android客戶端 |
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西門吹雪170: 金幣+3, 鼓勵回帖交流 2020-03-11 08:06:52
西門吹雪170: 金幣+3, 鼓勵回帖交流 2020-03-11 08:06:52
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Preparation of the cells: Day 1: Inoculate 5 ml of G/L-SGM17B medium with L. lactis glycerol stock from -80 C and grow at 30 C, without aeration, overnight Day 2: Inoculate 50 ml of G/L-SGM17B with pre-culture in a dilution of 1:100 and grow at 30 C, without aeration, overnight Day 3: - Add 50 ml full-grown culture to 400 ml of G/L-SGM17B medium - Grow the culture until OD600 is 0.2-0.3 (ca. 3 h) - Spin down cells for 20 min at 6000 x g, 4 C - Wash cells with 400 ml of 0.5 M sucrose, 10% glycerol (4 C) and spin down at 6000 x g (centrifugation speed may need to be increased during successive washing steps) - Resuspend the cells in 200 ml of 0.5 M sucrose, 10% glycerol, 50 mM EDTA (4 C), keep the suspension on ice for 15 min and spin down - Wash cells with 100 ml of 0.5 M sucrose, 10% glycerol (4 C) and spin down (6000 x g) - Resuspend the cells in 4 ml of 0.5 M sucrose, 10% glycerol (4 C): Use 40 μl per electroporation (keep on ice) Or store the cells in small portions at -80 C, let them defreeze on ice before use Electroporation: - Place 40 μl cells in a pre-chilled electroporation cuvette with 1 μl DNA (100-500 ng vector DNA reconstituted in TE-, Tris-buffer, or distilled water; for transforming cells with ligation product use 500-1000 ng DNA) and keep the cuvette on ice - Use Biorad Genepulser with following adjustments: 2000 V 25 μF 200 Ω - Pulse (normal reading is 4.5-5 msec) - Add 1 ml of G/L-M17B + 20 mM MgCl2 + 2 mM CaCl2 - Keep the cuvette for 5 min on ice and incubate 1-1.5 h at 30 C - Plate 10 μl, 100 μl, 900 μl on M17agar with glucose or lactose and antibiotics (depends on plasmid) - Incubate 1-2 days at 30 °C Materials: - G/L-SGM17B: M17-Broth with: 0.5 M sucrose 2.5% glycine 0.5% glucose or 0.5% lactose (strain dependent) Add the sucrose and glycine to the M17-B and sterilize 20 min 121 °C. Add sterile glucose or lactose after cooling down. - 0.5 M sucrose/ 10% glycerol - 0.5 M sucrose/ 10% glycerol/ 0.05 M EDTA L. lactis grows very slowly on G/L-SGM17B. Leaving out the sucrose is possible (Wells et al., 1993) but can decrease the transformation efficiency. The medium for cell recovery must contain MgCl2 and CaCl2. 我用這個protocol做的，效果還不錯 |
5樓2020-03-10 11:17:16
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