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金黃色葡萄球菌菌株基本信息
| 出品公司: | -- |
|---|---|
| 感受態細胞名稱: | RN4220 |
| 菌株類型: | 金黃色葡萄球菌 Staphylococcus aureus |
| 產品目錄號: | -- |
| 培養基: | MH 培養基 |
| 生長條件: | 37 ℃, 有氧 |
| 基因型: | |
| 抗性: | 無抗 |
| 質粒轉化條件: | 電轉 |
| 應用: | 金黃色葡萄球菌基因操作 |
| 誘導方法: | -- |
| 菌株特點: | RN4220一直以來被廣泛的應用于金葡菌基因研究。該菌株來源于NCTC8325-4，是一株限制性內切酶缺陷菌株，能夠接受來源于外部的其他物種的DNA質粒。RN4220體內沒有其他質粒，對大部分抗生素都敏感。 Electroporation of Staphylococcus aureus (Hooper lab protocol) 1. Grow cells overnight in Brain Heart Infusion Broth (BHI). 3-5ml culture is sufficient. 2. Add 1ml of overnight culture to 100ml fresh BHI. (OD550 should be ~ 0.010 at this point). Grow at 37℃ (shaker table) until OD550 is 0.2-0.25. Usually takes 2-2? hours for BHI. (Note: Check OD initially after 2 hours. For rapid growers like RN4220, it may already be at or near 0.2. For other strains, it’s often in the 0.12-0.15 range. Recheck every 15 minutes until culture reaches the target OD. Electroporation efficiency drops off quite markedly once OD550 > 0.3). 3. Wash 4x with ice cold 0.5M sucrose (17% v/v). Keep cold at this point. Go from 100ml to 25ml to 10ml to 1ml. Can use the table-top 4℃ Sorvall RT6000B centrifuge set at 9 for 3000 rpm. Resuspend final pellet in ~500ul of 0.5M sucrose. 4. Add 160ul of cells to DNA. Incubate on ice for 15 minutes. Don’t use more than 20?l of DNA in sterile water. (With an new plasmid prep, reasonable to try 5ul, 10ul, and 20ul of a Qiagen MidiPrep of ~100-300ng/ul vector DNA.) Concentrate DNA if need be. (Otherwise, you’re at risk for arcing). 5. Electroporate. Settings = 2.5kV, 200 Ohms, and 25uF. 6. Add 1ml BHI to cuvette, mix gently and transfer to sterile eppendorf tube, incubate at 30C 2 hours (30C because plasmids can be unstable at 37C in RN4220 independent of temp-sensitive origin of replication), and plate (50-200 ul of 1ml suspension) on appropriate antibiotic-selective BHI plates. Notes BHI is better than trypticase soy broth (TSB). Use BioRad Gene Pulsar with 2mm cuvettes. For chloramphenical, use 10ug/ml in plate. For erythromycin, use 3-10ug/ml in plate. For tetracycline, use 5ug/ml in plate. The time constant for electroporation should be about 4.2-4.6 msec. The cells can be frozen at –70C and used later but this will decrease the efficiency of transfer. (I suspect that there’s a lot of cell death without glycerol in the media). For 500ml 0.5M sucrose (MW 342.3 g/mole), add 85.6G sucrose in 500ml deionized water and filter sterilize (don’t autoclave). Store at 4℃. |
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金黃色葡萄球菌菌株應用舉例
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