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基 因 型Δ(ara-leu)7697ΔlacX74phoAPvuIIphoRaraD139ahpCgalEgalKrpsL(DE3)F[lac+lacIqpro]gor522::Tn10 trxB pLysSRARE2 (CamR , StrR , TetR )簡 要 說 明Rosetta-gami(DE3)pLysS 菌株聚合了不同原核表達(dá)菌株的優(yōu)勢：?Rosetta-gami賦予其 Rosetta和Origami的優(yōu)點——補(bǔ)充大腸桿菌缺乏的6 種稀有密碼子(AUA， AGG， AGA， CUA，CCC， GGA) 對應(yīng)的tRNA，提高外源基因的表達(dá)水平，并且包含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，表達(dá)主要還原途徑的兩個關(guān)鍵酶。有利于形成正確折疊的含有二硫鍵的蛋白，增強(qiáng)蛋白的可溶性。?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啟動子誘導(dǎo)的蛋白表達(dá)。?該菌株攜帶的pLysS質(zhì)粒含有表達(dá) T7 溶菌酶的基因，能夠降低目的基因的背景表達(dá)水平，但不干擾IPTG誘導(dǎo)的表達(dá)，適合表達(dá)毒性蛋白和非毒性蛋白。Rosetta-gami(DE3)pLysS 菌株具有卡那霉素，氯霉素，鏈霉素，四環(huán)素抗性，經(jīng)pUC19質(zhì)粒檢測轉(zhuǎn)化效率高達(dá)108cfu/μg。操 作 說 明1.取100μl冰上融化的Rosetta-gami(DE3)pLysS 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回冰上并靜置2分鐘，晃動會降低轉(zhuǎn)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(fù)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(yīng)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(xì)胞最好在冰上融化。2.混入質(zhì)粒時應(yīng)輕柔操作。3.轉(zhuǎn)化高濃度的質(zhì)?？上鄳?yīng)減少最終用于涂板的菌量。4.誘導(dǎo)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(dǎo)時間，溫度，IPTG濃度需實驗者優(yōu)化。6.菌株攜帶 pLysS質(zhì)粒，除復(fù)蘇培養(yǎng)基為無抗生素外，其余所用培養(yǎng)基、培養(yǎng)液均應(yīng)含有34 µg/ml氯霉素，以防質(zhì)粒丟失。7.具有卡那霉素抗性，不能用于具有卡那霉素抗性質(zhì)粒的表達(dá)。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). 

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