介绍： 基 因 型F- ompT hsdSB(rB- mB- ) gal dcm(DE3)简 要 说 明High5TM系列 BL21（DE3）感受态细胞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BL21的染色体上，所以称为BL21(DE3)。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。High5TM系列BL21（DE3） 感受态细胞由特殊工艺制作，经pUC19质粒检测转化效率达108cfu/μg。操 作 说 明1.取100μl冰上融化的BL21（DE3）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90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。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 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 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 101.minutes at 4℃.9.Remove the supernatant and store the cell pellet at -20℃ (storage at lower temperatures is also acceptable). IPTGPrepare 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.
新浪微博
腾讯微博
QQ空间
客服
