Tuner(DE3)Chemically Competent Cell

 基因型

F- ompT hsdSB(rB

-

mB

-

) gal dcm lacY1 (DE3)

● 产品说明

Tuner(DE3)菌株是 BL21 菌株的 lacZY 基因 (半乳糖苷透性酶基因)突变株，此突变导致 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。此菌株用于高效表达克隆于含有噬菌

体 T7 启动子的表达载体 (如 pET 系列)的基因。λ 噬菌体 DE3 区含有 T7 噬菌体 RNA 聚合酶，该区整合

于 Tuner 的染色体上，所以称为 Tuner(DE3)，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。Tuner(DE3)感受态细胞由特殊工艺制作，pUC19 质粒检

测转化效率达 107 cfu/μg DNA。

● 操作方法

1. Tuner(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 700 μl 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. 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 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 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

acceptable).

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. 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（0.1-2 mM 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

本产品仅供科研使用.请勿用于医药,不能用于临床治疗诊断使用！