HT115(DE3) Chemically Competent Cell 產品說明(ming)書
產品規格 （CAT#: EC2010）
HT115(DE3)： 100μl/支
pUC19 (control vector，10pg/μl): 10μl
保存條(tiao)件(保質(zhi)期)： -80℃（6個(ge)月(yue)）
基(ji)因型
F- mcrA mcrB, IN(rrnD-rrnE)1 rnc14::Tn10(DE3 lavUV5::T7 polymerase)
產品說(shuo)明
HT115(DE3)菌株是一(yi)類特(te)殊的RNase III缺陷型大(da)腸桿菌菌株，可(ke)以飼喂線(xian)蟲(chong)，主要用(yong)于秀麗隱(yin)桿線(xian)蟲(chong)（C. elegans）的RNAi干(gan)擾試(shi)驗(yan)。該菌株可(ke)以在(zai)LB或2YT培養基(ji)中(zhong)正常生長，Tn10轉(zhuan)(zhuan)座子的存在(zai)使(shi)其(qi)具(ju)有四環素抗性。該菌株染色體(ti)整合(he)了(le)(le)λ噬菌體(ti)DE3區 (DE3區含有T7噬菌體(ti)RNA聚(ju)合(he)酶(mei)，在(zai)IPTG存在(zai)時(shi)可(ke)誘(you)導T7 RNA聚(ju)合(he)酶(mei)大(da)量(liang)表(biao)(biao)達，進(jin)而啟動線(xian)蟲(chong)ds RNA的表(biao)(biao)達)，可(ke)同時(shi)表(biao)(biao)達T7 RNA聚(ju)合(he)酶(mei)和大(da)腸桿菌RNA聚(ju)合(he)酶(mei)，除了(le)(le)用(yong)于線(xian)蟲(chong)RNAi干(gan)擾試(shi)驗(yan)，也(ye)可(ke)用(yong)于pET系列(lie)，pGEX，pMAL等質粒的蛋(dan)白表(biao)(biao)達。HT115(DE3)感受態細(xi)胞由特殊工(gong)藝(yi)制作(zuo)，pUC19質粒檢測轉(zhuan)(zhuan)化效率達0.5×108 cfu/μg DNA。
操作方法
1. HT115(DE3)感(gan)受態細(xi)胞從-80℃拿(na)出，迅速插入(ru)冰(bing)(bing)中，5分(fen)鐘后待(dai)菌塊融化，加入(ru)目的質(zhi)粒并用手撥打(da)EP管(guan)底(di)輕輕混(hun)勻(避免用槍(qiang)吸打(da))，冰(bing)(bing)中靜置25分(fen)鐘。
2. 42℃水(shui)浴(yu)熱激45秒，迅速放回冰中并靜(jing)置2分鐘，晃動會降低(di)轉化效率。
3. 向(xiang)離心管(guan)中加入700 μl不含抗生素的LB無菌培養液(ye)，37℃，200 rpm復蘇60分鐘。
4. 5000 rpm離(li)心一分鐘收(shou)菌(jun)，留(liu)取(qu)50 μl左右(you)上清輕(qing)輕(qing)吹打重懸菌(jun)塊并涂布到(dao)含相應抗生素的LB培(pei)養基上。
5. 將(jiang)平板倒置放于37℃培(pei)養箱過夜培(pei)養。
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.
注意事項
1. 感(gan)受態細(xi)胞最好在冰(bing)(bing)中緩慢融(rong)化，插入冰(bing)(bing)中8分鐘(zhong)內(nei)加入目標DNA，不(bu)可在冰(bing)(bing)中放(fang)置(zhi)時間過長，長時間存放(fang)會降低轉(zhuan)化效(xiao)率。
2. 轉化高濃度的質粒(li)可(ke)相應減少最(zui)終用(yong)于涂板的菌量。
3. 誘導(dao)時，IPTG濃度可根據實驗選擇使(shi)用（0.1-10mM）。
4. 進行線蟲RNAi質(zhi)粒轉(zhuan)化時，抗(kang)生(sheng)素(su)使用濃(nong)度可參考：氨芐—50ug/ml，四環素(su)—12.5ug/ml。
5. 若進行蛋(dan)白表(biao)達，為(wei)獲(huo)得需要量(liang)的蛋(dan)白，最佳(jia)誘導時間，溫度(du)，IPTG濃(nong)度(du)需實驗者優化。
