GV3101（pSoup）Electroporation-Competent Cell

GV3101（pSoup）Electroporation-Competent Cell

运输与保存方法：干冰运输

保存温度：-80°保存

操作说明

1. 取 100μl 冰上融化的 GV3101（pSoup）Electroporation-Competent Cell感受态细胞，加入目的质粒并轻轻混匀，冰上 静置 25 分钟。GV3101（pSoup）Electroporation-Competent Cell10×100μl pUC19（control vector） 10pg/μl

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。

注意事项：

1. 感受态细胞在冰上融化。

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，诱导时间，温度，IPTG 浓度需实验者优化。 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 bebetter).

 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 untilneeded for gel analysis or storage at -20℃. These will serve as the non-induced controlsamples.

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