| 出品公司: | -- |
|---|---|
| 感受态细胞名称: | RN4220 |
| 菌株类型: | 金黄色葡萄球菌 Staphylococcus aureus |
| 产品目录号: | -- |
| 培养基: | MH 培养基 |
| 生长条件: | 37 ℃, 有氧 |
| 基因型: | |
| 抗性: | 无抗 |
| 质粒转化条件: | 电转 |
| 应用: | 金黄色葡萄球菌基因操作 |
| 诱导方法: | -- |
| 菌株特点: | RN4220一直以来被广泛的应用于金葡菌基因研究。该菌株来源于NCTC8325-4，是一株限制性内切酶缺陷菌株，能够接受来源于外部的其他物种的DNA质粒。RN4220体内没有其他质粒，对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 2-2½ 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 20l 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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