产品介绍

基 因 型Δ(ara-leu)7697ΔlacX74phoAPvuIIphoRaraD139ahpCgalEgalKrpsL(DE3)F[lac+lacIqpro]gor522::Tn10 trxB pLysSRARE2 (CamR , StrR , TetR )简 要 说 明Rosetta-gami(DE3)pLysS 菌株缔合了不同于原核表现菌株的优势与劣势：Rosetta-gami拥有其 Rosetta和Origami的的优势——多补肠子杆菌由于缺乏的6 种难得支付密码子(AUA， AGG， AGA， CUA，CCC， GGA) 相对应的的tRNA，增进外源dna的表现情况，然后涉及到固色体突变的硫氧还核血清呈现酶（thioredoxin reductase）(trxB) 和谷胱甘肽呈现酶(glutathione reductase)(gor)dna，表现通常呈现方式的两人根本酶。重要于进行规范折起来的含二硫键的核血清，怎强核血清的可阴离子型。该菌株固色体优化系统组合了λ噬菌体DE3区（DE3区含T7噬菌体RNA缔合酶），满足T7启动的子诱惑的核血清表现。该菌株过飞机安检的pLysS质粒含表现 T7 溶菌酶的dna，还可以调低从而为的dna的经验表现情况，但不电磁波辐射IPTG诱惑的表现，满足表现渗透性核血清和非渗透性核血清。Rosetta-gami(DE3)pLysS 菌株带有卡那霉素，氯霉素，链霉素，四环素抗性，经pUC19质粒探测转变成学习速率高至108cfu/μg。操 作 说 明1.取100μl冰面运动溶解的Rosetta-gami(DE3)pLysS 体会心得态人体肿瘤细胞，引入从而为的质粒并温柔地混匀，冰面运动静置24秒钟。2.42回冰面运动并静置2秒钟，摆动会调低转变成学习速率。3.向抽滤法管内引入700μl不标四环素的无菌检测锻炼出基（2YT或LB），混匀后37℃，200rpm溶栓60秒钟。4.5000rpm抽滤法一秒钟收菌，留取100μl影响上清温柔地吹打重悬菌块并涂覆到含某些四环素的2YT 或LB锻炼出基上。5.将平板电脑倒放放于37℃锻炼出箱過夜锻炼出。注 意 事 项1. 体会心得态人体肿瘤细胞尽量在冰面运动溶解。2.吸进质粒时先温柔基本操作。3.转变成高密度的质粒可某些削减从而使中用涂板的菌量。4.诱惑时，IPTG密度可供选择（0.1-2mM均可）。5.为收获都要量的核血清，合适诱惑时刻，室内温度，IPTG密度需实验报告者优化系统。6.菌株过飞机安检 pLysS质粒，除溶栓锻炼出基为无四环素外，任意所有锻炼出基、锻炼出液均应含34 µg/ml氯霉素，为防质粒流失。7.带有卡那霉素抗性，没能使中用带有卡那霉素抗成分粒的表现。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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