BL21 Star(DE3)感受态细胞说明书

  • 来源:上海钰博生物科技有限公司
  • 时间: 2018/12/3
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     BL21 Star(DE3)感受态细胞说明书 

    BL21 Star(DE3):                                        100μl/支

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

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

     


    基因型

    FompT hsdSB(rBmBgal dcm rne131 (DE3)


    产品说明

    BL21 Star(DE3)菌株源于BL21(DE3)菌株,含有rne131突变(RNaseE基因),RNaseE基因的突变降低了內源RNase的积累,增强菌株细胞内mRNA的稳定性,从而提高异源蛋白的表达水平。主要适用于T7启动子表达载体(如pET系列)的高水平蛋白表达,同时含有大肠杆菌RNA聚合酶,也可用于非T7启动子表达载体(pGEX,pMAL等)的蛋白表达。由于BL21 Star(DE3)菌株的异源基因基础表达水平较高,所以不适合毒性蛋白的表达。BL21 Star(DE3)感受态细胞由特殊工艺制作,pUC19质粒检测转化效率达108cfu/μg DNA。


    操作方法

    1.  BL21 Star(DE3) 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(质粒或连接产物)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(2YTLB),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相应抗生素的2YTLB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 min 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。


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