BL21(DE3)感受態(tài)細(xì)胞使用說(shuō)明

1、北京華越洋生物科技有限公司HUAYUEYANG BIOTECHNOLOGY CBEIJING) CO. LTDBL21(DE3感受態(tài)細(xì)胞產(chǎn)品編碼產(chǎn)品名稱規(guī)格WR105-100SBL21(DE3感受態(tài)細(xì)胞10*100ulWR105-100BL21(DE3感受態(tài)細(xì)胞20*100BL21(DE3感受態(tài)細(xì)胞簡(jiǎn)介：BL21 ( DE3)感受態(tài)細(xì)胞是采用大腸桿菌 BL21 ( DE3)菌株經(jīng) 特殊工藝處理得到的感受態(tài)細(xì)胞，可用于DNA的化學(xué)轉(zhuǎn)化。使用 pUC19質(zhì)粒檢測(cè)，轉(zhuǎn)化效率可達(dá)107, -70C保存幾個(gè)月轉(zhuǎn) 化效率不發(fā)生改變。BL21(DE3菌株適合表達(dá)非毒性蛋白。該菌株是以T7 RNA聚合酶為表達(dá)

2、系統(tǒng)的外源基因蛋白高效表達(dá)的宿主。T7噬菌體RNA聚合酶基因的表達(dá)受 入噬菌體DE3區(qū)的lacUV5啟動(dòng)子 調(diào)控，該區(qū)整合在 BL21染色體上。本產(chǎn)品是大腸桿菌BL21(DE3菌株經(jīng)特殊工藝處理得到的感受態(tài)細(xì)胞，可用于DNA的熱擊轉(zhuǎn)化。使用 pU19持粒檢測(cè)，轉(zhuǎn)化效率可達(dá)107。BL21(DE3感受態(tài)細(xì)胞產(chǎn)品特點(diǎn)轉(zhuǎn)化效率可達(dá)107。可用于非毒性蛋白表達(dá)。長(zhǎng)時(shí)間保存于-80 C,轉(zhuǎn)化效率不發(fā)生改變。保存條件：-80 CBL21(DE3感受態(tài)細(xì)胞基因型-_-+ r rF ompT hsdS(rB mB ) dcm Tet gal 入(DE3) endA Hte argU proL Cam argU

3、 ileY leuW rStrep/Spec BL21(DE3感受態(tài)細(xì)胞說(shuō)明BL21-CodonPlus(DE3)-RIPL菌株來(lái)源于 Stratagene 公司的 BL21-Gold 菌株，缺少 Lon 蛋白酶和 OmpT蛋白酶，從而減少對(duì)重組蛋白的降解，補(bǔ)充大腸桿菌缺乏的4種稀有密碼子(AGA、AUA、CCC CUA)對(duì)應(yīng)的tRNA ( argU、ileY、proL、leuW),提高外源基因，尤其是富含AT-或GC的真核基因在原核系統(tǒng)中的表達(dá)水平。該菌株染色體整合了入噬菌體DE3區(qū)(DE3區(qū)含有T7噬菌體RNA聚合酶)，可同時(shí)表達(dá) T7 RNA聚合酶和大腸桿菌 RNA聚合酶，可用于 pET

4、系列，pGEX pMAL等質(zhì)粒的蛋白表達(dá)，同時(shí)具有四環(huán)素，氯霉素，鏈霉素，壯觀霉素 抗性。High5TM系列BL21- Codon Plus(DE3)-RIPI感受態(tài)細(xì)胞由特殊工藝制作，經(jīng)pUC19質(zhì)粒檢測(cè)轉(zhuǎn)化效率高達(dá)108cfu/ goBL21(DE3感受態(tài)細(xì)胞操作方法1. 取100 1冰上融化的 BL21-Codo nPlus(DE3)-RIPL感受態(tài)細(xì)胞，加入目的質(zhì)粒并輕輕混勻，冰上靜置25分鐘。2. 42 C水浴熱激45秒，迅速放回冰上并靜置2分鐘，晃動(dòng)會(huì)降低轉(zhuǎn)化效率。3. 向離心管中加入 700 1不含抗生素的無(wú)菌培養(yǎng)基(2YT或LB),混勻后37C, 200rpm復(fù)蘇60分鐘。4.

5、 5000rpm離心一分鐘收菌，留取100卩1右上清輕輕吹打重懸菌塊并涂布到含相應(yīng)抗生素 的2YT或LB培 養(yǎng)基上。北京華越洋生物科技有限公司HUAYUEYANG BIOTECHNOLOGY CBERING) CO. LTD5. 將平板倒置放于37 C培養(yǎng)箱過(guò)夜培養(yǎng)。Sample Induction Protocol (or referenee only )1. Ino eulate a sin gle colony from a freshly streaked plate into 5 ml of LB medium containing the appropriate antibioti

6、c for the plasmid and host strain.2. Incubate with shaking at 200 rpm at 37 °C overnight.3. I noculate 50 ml of LB medium containing the appropriate antibiotic with 0.5 ml of the overni ght culture prepared in step 2(use the 500 ml tria ngular flask as the container would be better).4. Incubate

7、 with shaking at 150 rpm at 37 C 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 iceuntiIneeded for gel analysis or storage at -20 C . These will serve as the non-induced control samples.6. Add IPTG to a final concen tra

8、tio n of 1 mM. Optimal time for in duct ion of the target protein may vary from 2-16 hours, depe nding on the prote in.7. Incubate with shaking at 120 rpm at 37 C for 3-4 hours. To determine the optimal time for in duct ion of the target prote in, it is recomme nded that a time course experime nt be

9、 performedvary ing the in duct ion from 2-16 hours.8. Place the culture on ice for 10 minu tes. Harvest cells by cen trifugati on at 5,000 x g for 10 minutes at 4 C .9. Remove the super nata nt and store the cell pellet at -20 C (storage at lower temperatures is also acceptable).IPTGPrepare a 1 M soluti on of IPTG (Isopropyl- 3 -D-thiogalactoside; Isopropyl- 3 -D-thiogalactopyra no side) bydissolving 2.38 g of IPTG in dd water and adjust the final volume to 10 ml.