ArcticExpress (DE3) pRARE2感受态细胞说明书

  • 来源:上海钰博生物科技有限公司
  • 时间: 2018/11/21
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     ArcticExpress (DE3) pRARE2感受态细胞说明书 

    产品规格 

     

    ArcticExpress (DE3) pRARE2:                         100μl/支

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

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

     


    基因型


    E. coli B F– ompT hsdS(rB– mB–) dcm+ TetR gal λ(DE3) endA Hte [cpn10cpn60 GentR] pRARE2 (CamR)

     

     

    产品说明

     

    ArcticExpress (DE3) pRARE2来源于ArcticExpress (DE3),将具有氯霉素抗性的pRARE2质粒导入ArcticExpress (DE3)细胞中即是ArcticExpress (DE3) pRARE2。ArcticExpress (DE3) pRARE2菌株染色体DNA中整合了λ噬菌体DE3区,使得ArcticExpress (DE3) pRARE2菌株可同时表达T7 RNA聚合酶和大肠杆菌RNA聚合酶,广泛用于pET系列,pGEX,pMAL等质粒的蛋白表达。ArcticExpress (DE3) pRARE2菌株具有四环素,庆大霉素,氯霉素抗性,endA1突变有利于质粒DNA的稳定。[cpn10cpn60 GentR]的存在使ArcticExpress (DE3) pRARE2可以表达适应低温的伴侣蛋白Cpn10和Cpn60(来自嗜冷菌—Oleispira antarctica)。Cpn10和Cpn60伴侣蛋白在4-12℃表现出较高活性,在ArcticExpress(DE3) pRARE2细胞中表达时,可降低重组蛋白包涵体的形成,增加可溶重组蛋白的表达量及生物活性,比传统的原核表达伴侣蛋白GroEL、GroES等具有更加优异的促融能力。同时,pRARE2质粒可补充大肠杆菌缺乏的7种稀有密码子(AUA, AGG, AGA, CUA, CCC, GGA和CGG)对应的tRNA,提高外源基因的表达水平。唯地生物生产的ArcticExpress (DE3) pRARE2感受态细胞经特殊工艺制作,pUC19质粒检测转化效率达108cfu/μg DNA。

     

    操作方法

    1. ArcticExpress (DE3) pRARE2感受态细胞从-80℃拿出,迅速插入冰中,5分钟后待菌块融化,加入目的质粒,并用手拨打EP管底混匀,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700 μl不含抗生素的无菌培养基 (LB),混匀后37℃,200 rpm复苏60分钟。

    4. 5000 rpm离心一分钟收菌,留取100 μl左右上清轻轻吹打重悬菌块并涂布到含相应抗生素的LB培养基上((平板中务必同时含有34ug/ml的氯霉素,40ug/ml的庆大霉素和转化质粒本身的筛选抗生素;若质粒浓度较高,也可稀释后涂板,务必保证能在平板上挑到单克隆菌落)。

    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 3ml 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. (0.6 recommended; about 2.5h).

    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 2-4 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 accep- table).  

     

     

     

     

    IPTG配制:

         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. ArcticExpress (DE3) pRARE2菌株携带 pRARE2质粒,除复苏培养基为无抗生素外,其余所用培养基、培养液均应含有34 µg/ml氯霉素、40ug/ml的庆大霉素,以防质粒丢失。

    3. 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。

     

     

     

    4. ArcticExpress (DE3) pRARE2感受态细胞具有四环素、庆大霉素、氯霉素抗性,不可用于具有四环素、庆大霉素、氯霉素抗性质粒的转化。

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