OverExpress C43(DE3) 热敏感受态细胞

OverExpress C43(DE3) Chemically Competent Cell

产品规格:

OverExpress C43(DE3) 10×100μl

pUC19(control vector) 10pg/μl 10μl

基 因 型

F–ompT hsdSB (rB- mB-) gal dcm (DE3)OverExpress C43(DE3)、OverExpress C41(DE3)两个菌株均起源于 BL21(DE3)，其优点是可以高效表达毒性蛋白或

疏水性蛋白。OverExpress C41(DE3)跟 BL21(DE3)的区别在于其基因组含有至少一个未知突变，这个未知突变使其获得了高效表达

毒性蛋白 (1-5)的能力，此突变位点参与大肠杆菌表达毒性蛋白时的细胞死亡途径；OverExpress C43(DE3)来源于

OverExpress C41(DE3)，是通过筛选 OverExpress C41(DE3)对另一个不同毒性蛋白的抗性菌株获得。 OverExpress C43(DE3)菌株具有比 OverExpress C41(DE3)更强的表达毒性蛋白和疏水性蛋白的能力，所以说

OverExpress C43(DE3) 菌株与 BL21(DE3)相比拥有至少两个未知突变，正是这两个未知突变使其获得了更广泛的表

简 要 说 明

达毒性蛋白或疏水性蛋白的能力。此菌株含有 DE3 区，可同时表达 T7 RNA 聚合酶和大肠杆菌 RNA 聚合酶，可用于

pET 系列，pGEX，pMAL 等质粒的蛋白表达。OverExpress C43(DE3)感受态细胞由特殊工艺制作，pUC19 质粒检测转化效率可达 5×108 cfu/μg

DNA。

1. OverExpress C43(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 (2YT 或 LB)，混匀后 37℃，200 rpm 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 2YT 或 LB 培养基

上。

5. 将平板倒置放于 37℃培养箱过夜培养。

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.

操 作 说 明

Sample Induction Protocol (for reference only)

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.

1.感受态细胞最好在冰上融化。 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.

产品优势

1、蛋白表达：适合表达非毒性蛋白

2、效率高：可达到 107cfu/ug pUC19 DNA

3、稳定性好：-70 度冰箱可保存 6 个月时间

保存条件： -80℃