產(chǎn)品介紹

基 因 型D(ara-leu)7697 DlacX74 DphoA PvuII phoR araD139 ahpC galE galK rpsL (DE3) F’[lac+ lacIq pro] gor522::Tn10 trxB pRARE2 (CamR, StrR, TetR)簡 要 說 明Rosetta-gami 2(DE3) 菌株集合了Rosetta 2 和 Origami 2 兩種菌株的優(yōu)點(diǎn)： pRARE2賦予其Rosetta2菌株的優(yōu)點(diǎn)——補(bǔ)充大腸桿菌缺乏的7種稀有密碼子(AUA, AGG, AGA, CUA, CCC, GGA和CGG)對應(yīng)的tRNA，提高外源基因的表達(dá)水平。 gor522::Tn10 trxB賦予其Origami2菌株的優(yōu)點(diǎn)——突變的硫氧還蛋白還原酶(thioredoxin reductase) (trxB)和谷胱甘肽還原酶(glutathione reductase) (gor)基因，它們是還原途徑的兩個(gè)關(guān)鍵酶，其突變有利于高效形成正確折疊的含有二硫鍵的蛋白，增強(qiáng)蛋白的可溶性；同時(shí)該菌株不具有卡那霉素抗性，可用于具有卡那霉素抗性質(zhì)粒的蛋白表達(dá)。該菌株染色體整合了λ噬菌體DE3區(qū) (DE3區(qū)含有T7噬菌體RNA聚合酶)適合T7啟動(dòng)子誘導(dǎo)的蛋白表達(dá)。Rosetta-gamiB(DE3)菌株具有氯霉素，鏈霉素，四環(huán)素抗性，由特殊工藝制作，經(jīng)pUC19質(zhì)粒檢測轉(zhuǎn)化效率高達(dá)108 cfu/μg DNA。操 作 說 明1.Rosetta-gami 2(DE3)菌株感受態(tài)細(xì)胞從-80℃拿出，迅速插入冰中，5分鐘后待菌塊融化，加入目的DNA并用手撥打EP管底輕輕混勻(避免用槍吸打)，冰中靜置25分鐘。2.42℃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 μl不含抗生素的無菌培養(yǎng)基 (2YT或LB)，混勻后37℃，200 rpm復(fù)蘇60分鐘。4.5000 rpm離心一分鐘收菌，留取100 μl左右上清輕輕吹打重懸菌塊并涂布到含所選質(zhì)粒篩選抗生素的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(xiàng)1.感受態(tài)細(xì)胞最好在冰中緩慢融化，插入冰中8分鐘內(nèi)加入目標(biāo)DNA，不可在冰中放置時(shí)間過長，長時(shí)間存放會(huì)降低轉(zhuǎn)化效率。 2.混入質(zhì)粒時(shí)應(yīng)輕柔操作。 3.轉(zhuǎn)化高濃度的質(zhì)粒可相應(yīng)減少最終用于涂板的菌量。 4.誘導(dǎo)時(shí)，IPTG濃度可選（0.1-2 mM均可）。 5.為獲得需要量的蛋白，最佳誘導(dǎo)時(shí)間，溫度，IPTG濃度需實(shí)驗(yàn)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×g for 10 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) by dissolving 2.38 g of IPTG in dd water and adjust the final volume to 10 ml. Filter sterilize before use.

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