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
菌株来源: 金黄色葡萄球菌RN4220菌株,是利用紫外线UV和化学方法,诱导NCTC8325菌株产生的突变株。
产品目录号: BF4220
培养基: MH 培养基,TSB培养基,Brain heart infusion Broth培养基
生长条件: 37 ℃,  有氧
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: --

金葡菌RN4220是用来转化大肠杆菌质粒DNA的金黄色葡萄球菌菌株。该菌株在基因sau1 hsdR上有突变,该突变的产生可以导致RN4220成为限制修饰系统基因缺陷型,可以用来作为大肠杆菌质粒DNA和金黄色葡萄球菌质粒转化的中间宿主菌。该菌株基因型是mec阴性,rsbU阴性,agr阴性。MLST测序分型为8,e基因组SPA分型为59,e基因组SPA重复序列为YHGGFMBQBLO。 Ridom SPA分型为t211。RN4220的基因组序列已经测序成功,Genbank编号为AFGU00000000.1。

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 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.

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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