產品介紹

基 因 型E. coli B F- dcm ompT hsdS(rB- mB-) gal [malB+]K-12(λS)簡 要 說 明BL21是最早開發(fā)的用于原核表達的菌株，BL21（DE3）、Rosetta、OrigamiB(DE3) 等一系列原核表達菌株均來源于BL21菌株。該菌株主要用于非毒性蛋白的表達，不含T7 RNA聚合酶，所以不能用于由T7啟動子驅動的蛋白表達(如：pET系列)；但含有大腸桿菌RNA聚合酶，可以用于tac或trc等使用大腸桿菌RNA聚合酶的原核系統(tǒng)的表達（如：pGEX，pMAL質粒）。High5TM系列 BL21感受態(tài)細胞由特殊工藝制作，經pUC19質粒檢測轉化效率達107cfu/μg。操 作 說 明1.取100μl冰上融化的BL21感受態(tài)細胞，加入目的質粒并輕輕混勻，冰上靜置25分鐘。2.42℃水浴熱激90秒，迅速放回冰上并靜置2分鐘，晃動會降低轉化效率。3.向離心管中加入700μl不含抗生素的無菌培養(yǎng)基（2YT或LB），混勻后37℃，200rpm復蘇60分鐘。4.5000rpm離心一分鐘收菌，留取100μl左右上清輕輕吹打重懸菌塊并涂布到含相應抗生素的2YT或LB培養(yǎng)基上。5.將平板倒置放于37℃培養(yǎng)箱過夜培養(yǎng)。注 意 事 項1.感受態(tài)細胞最好在冰上融化。2.混入質粒時應輕柔操作。3.轉化高濃度的質粒可相應減少最終用于涂板的菌量。4.誘導時，IPTG濃度可選（0.1-2mM均可）。5.為獲得需要量的蛋白，最佳誘導時間，溫度，IPTG濃度需實驗者優(yōu)化。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 x g for 10minutes 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. 

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