HT115(DE3) Chemically Competent Cell 产品说明书
产品规格 （CAT#: EC2010）
HT115(DE3)： 100μl/支
pUC19 (control vector，10pg/μl): 10μl
保存条件(保质期)： -80℃（6个月）
基因型
F- mcrA mcrB, IN(rrnD-rrnE)1 rnc14::Tn10(DE3 lavUV5::T7 polymerase)
产品说明
HT115(DE3)菌株是一类特殊的RNase III缺陷型大肠杆菌菌株，可以饲喂线虫，主要用于秀丽隐杆线虫（C. elegans）的RNAi干扰试验。该菌株可以在LB或2YT培养基中正常生长，Tn10转座子的存在使其具有四环素抗性。该菌株染色体整合了λ噬菌体DE3区 (DE3区含有T7噬菌体RNA聚合酶，在IPTG存在时可诱导T7 RNA聚合酶大量表达，进而启动线虫ds RNA的表达)，可同时表达T7 RNA聚合酶和大肠杆菌RNA聚合酶，除了用于线虫RNAi干扰试验，也可用于pET系列，pGEX，pMAL等质粒的蛋白表达。HT115(DE3)感受态细胞由特殊工艺制作，pUC19质粒检测转化效率达0.5×108 cfu/μg DNA。
操作方法
1. HT115(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，迅速放回冰中并静置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LB无菌培养液，37℃，200 rpm复苏60分钟。
4. 5000 rpm离心一分钟收菌，留取50 μl左右上清轻轻吹打重悬菌块并涂布到含相应抗生素的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浓度可根据实验选择使用（0.1-10mM）。
4. 进行线虫RNAi质粒转化时，抗生素使用浓度可参考：氨苄—50ug/ml，四环素—12.5ug/ml。
5. 若进行蛋白表达，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。
