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Rosetta-gami B(DE3)

文字:[大][中][小] 2016-3-31    浏览次数:3931    

Rosetta-gami B(DE3) Chemically Competent Cell 产品说明书




产品规格 (CAT#: EC1012)

Rosetta-gami B(DE3):                             100μl/支

pUC19 (control vector,10pg/μl):                    10μl

保存条件(保质期):                             -80℃(6个月) 


基因型

F- ompT hsdSB(rB-mB-) gal dcm lacY1 ahpC (DE3) gor522::Tn10 trxB pRARE (CamR, KanR, TetR)


产品说明

Rosetta-gami B(DE3) 菌株聚合了BL21,Tuner,Origami和 Rosetta四种菌株的优点:

lacY1基因 (半乳糖苷透性酶基因)突变赋予其Tuner菌株的优点——IPTG以均一速度进入体系中大肠杆菌的每个细胞,产生更加严格、均一的浓度依赖。

pRARE赋予其Rosetta菌株的优点——补充大肠杆菌缺乏的6 种稀有密码子(AUA, AGG, AGA, CUA, CCC, GGA)对应的tRNA,提高外源基因的表达水平。

gor522::Tn10 trxB赋予其Origami菌株的优点——突变的硫氧还蛋白还原酶(thioredoxin reductase) (trxB)和谷胱甘肽还原酶(glutathione reductase) (gor)基因,它们是还原途径的两个关键酶,其突变有利于高效形成正确折叠的含有二硫键的蛋白,增强蛋白的可溶性。

该菌株染色体整合了λ噬菌体DE3区 (DE3区含有T7噬菌体RNA聚合酶)适合T7启动子诱导的蛋白表达。

Rosetta-gamiB(DE3)菌株具有卡那霉素,氯霉素,四环素抗性,由特殊工艺制作,经pUC19质粒检测转化效率高达108 cfu/μg DNA。

 

操作方法

1. Rosetta-gamiB(DE3)菌株感受态细胞从-80℃拿出,迅速插入冰中,5分钟后待菌块融化,加入目的DNA并用手拨打EP管底轻轻混匀(避免用枪吸打),冰中静置25分钟。

2. 42℃水浴热激45秒,迅速放回冰上并静置2分钟,晃动会降低转化效率。

3. 向离心管中加入700μl不含抗生素的无菌培养基 (2YT或LB),混匀后37℃,200rpm复苏60分钟。

4. 5000rpm离心一分钟收菌,,留取100μl左右上清轻轻吹打重悬菌块并涂布到含34 µg/ml氯霉素及所选质粒筛选抗生素的2YT 或LB培养基上。

5. 将平板倒置放于37℃培养箱过夜培养。


Sample Induction Protocol(for reference only)

1.   Inoculate a single colony from a freshly streaked plate into 5 ml of LB medium containing the appropriate antibiotic for the plasmid and host strain.

2.   Incubate with shaking at 200 rpm at 37℃ overnight.  

3.   Inoculate 50 ml of LB medium containing the appropriate antibiotic with 0.5 ml of the overnight culture prepared in step 2(use the 500 ml triangular flask as the container would be better).

4.   Incubate with shaking at 150 rpm at 37℃ until the OD 600 reaches 0.5-0.8.

5.   (Optional)Pipet 1ml of  the cultures into clean microcentrifuge tubes and  place the tubes on ice until  needed  for gel analysis or storage at  -20℃. These will serve as the non-induced control samples.      

6.   Add IPTG to a final concentration of 1 mM.  Optimal time for induction of the target protein may vary from 2-16 hours, depending on the protein.

7.   Incubate with shaking at 120 rpm at 37℃ for 3-4 hours. To determine the optimal time for induction of the target protein, it is recommended that a time course experiment be performed varying the induction from 2-16 hours.

8.   Place the culture on ice for 10 minutes. Harvest cells by centrifugation at 5,000×g for 10 minutes at 4℃.

9.   Remove the supernatant and store the cell pellet at -20℃ (storage at lower temperatures is also acceptable).  

IPTG

Prepare a 1 M solution of IPTG (Isopropyl-β-D-thiogalactoside; Isopropyl-β-D-thiogalactopyranoside) by

dissolving 2.38 g of IPTG in dd water and adjust the final volume to 10 ml. Filter sterilize before use.


注意事项

1. 感受态细胞最好在冰中缓慢融化,插入冰中8分钟内加入目标DNA,不可在冰中放置时间过长,长时间存放会降低转化效率。

2. 混入质粒时应轻柔操作。

3. 转化高浓度的质粒可相应减少最终用于涂板的菌量。

4. 诱导时,IPTG浓度可选(0.1-2mM均可)。

5. 为获得需要量的蛋白,最佳诱导时间,温度,IPTG浓度需实验者优化。

6. 具有卡那霉素抗性,不能用于具有卡那霉素抗性质粒的表达。






说明书下载

Rosetta-gami B(DE3) Chemically Competent Cell 产品说明书


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