BL21感受态细胞使用说明.docx
BL21感受态细胞使用说明 BL21(DE3)感受态细胞 产品编码 WR105-100S WR105-100 BL21(DE3)感受态细胞简介： BL21感受态细胞是采用大肠杆菌BL21菌株经特殊工艺处理得到的感受态细胞，可用于DNA的化学转化。使用pUC19质粒检测，转化效率可达107，-70保存几个月转 化效率不发生改变。 BL21(DE3)菌株适合表达非毒性蛋白。该菌株是以T7 RNA 聚合酶为表达系统的外源基因蛋白高效表达的宿主。T7 噬菌体RNA 聚合酶基因的表达受噬菌体DE3区的lacUV5 启动子调控，该区整合在BL21 染色体上。本产品是大肠杆菌BL21(DE3)菌株经特殊工艺处理得到的感受态细胞，可用于DNA的热击转化。使用pU19持粒检测，转化效率可达107。 BL21(DE3)感受态细胞产品特点 ·转化效率可达107。 ·可用于非毒性蛋白表达。 ·长时间保存于-80 ，转化效率不发生改变。 保存条件：-80 BL21(DE3)感受态细胞基因型 FompT hsdS(rB mB) dcm+ Tetr gal(DE3) endA Hte argU proL Camr argU ileY leuW Strep/Specr BL21(DE3)感受态细胞说明 BL21-CodonPlus(DE3)-RIPL菌株来源于Stratagene公司的BL21-Gold 菌株，缺少Lon蛋白酶和OmpT蛋白酶，从而减少对重组蛋白的降解，补充大肠杆菌缺乏的4种稀有密码子( AGA、AUA、CCC、CUA) 对应的tRNA ，提高外源基因，尤其是富含AT-或 GC-的真核基因在原核系统中的表达水平。该菌株染色体整合了噬菌体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，同时具有四环素，氯霉素，链霉素，壮观霉素抗性。High5TM系列BL21- Codon Plus(DE3)-RIPL 感受态细胞由特殊工艺制作，经pUC19质粒检测转化效率高达108cfu/g。 BL21(DE3)感受态细胞操作方法 1. 取100l冰上融化的BL21-CodonPlus(DE3)-RIPL感受态细胞，加入目的质粒并轻轻混匀，冰上静置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700l不含抗生素的无菌培养基，混匀后37，200rpm复苏60分钟。 4. 5000rpm离心一分钟收菌，留取100l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。 产品名称 BL21(DE3)感受态细胞 BL21(DE3)感受态细胞 规格 10*100ul 20*100 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 untilneeded 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 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-D-thiogalactoside; Isopropyl-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。 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。
