W3110(DE3)感受态细胞

W3110(DE3)感受态细胞、菌株，化转电转感受态细胞

　W3110(DE3) Chemically Competent Cell  CAT#: HYY7632

W3110(DE3) ：                                           100μl/支

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

基因型F- λ- rph-1 INV(rrnD, rrnE)(DE3)

产品说明W3110(DE3)是K-12的衍生菌株，是一种经过较少改造，比较接近于“WT-野生型”的大肠杆菌工程菌株，基因组与MG1655菌株高度相似（与MG1655基因组相比，有8个位点的基因突变）。W3110(DE3)菌株在液体培养时可耐受更高的菌体浓度，同时蛋白产量明显高于其他原核表达菌株，可作为蛋白表达的宿主菌株使用，提高菌体和蛋白产量。W3110(DE3)菌株染色体整合DE3区 (DE3区含有T7噬菌体RNA聚合酶)，该区整合于大肠杆菌的染色体上，可同时表达T7 RNA聚合酶和大肠杆菌RNA聚合酶，用于pET系列，pGEX，pMAL等质粒的蛋白表达，W3110(DE3)菌株不含核酸酶endA1突变，体内核酸酶含量较高，提取质粒时务必使用质粒提取试剂盒中去蛋白液尽量去除核酸酶，以防提取的质粒被降解。W3110(DE3)感受态细胞由特殊工艺制作，转化效率高。

操作方法

1. W3110(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，加入目的质粒并用手拨打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.  转化高浓度的质粒可相应减少最终用于涂板的菌量。

3. 诱导时，IPTG浓度可选（0.1-2 mM均可）。

4. 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。