产品介绍

基 因 型F- ompT hsdS(rB-mB-) gal dcm(DE3)pLysS Camr简 要 说 明High5TM国产表BL21(DE3)pLysS 感悟态上皮上皮体細胞系带入 pLysS质粒，还具有氯霉素抗性。pLysS包含展现 T7 溶菌酶的染色法体，T7溶菌酶能能用途于直肠杆菌上皮上皮体細胞系体两侧的肽聚糖熔化分解直肠杆菌，也能变低需求染色法体的时代背景展现水准，但不干预IPTG成脂的展现，可以展现致癌性蛋清和非致癌性蛋清。该菌株染色法体数据整合了λ噬菌体DE3区（DE3区包含T7噬菌体RNA聚合物酶），High5TM国产表BL21(DE3)pLysS 感悟态上皮上皮体細胞系由层次性工艺设计开发经pUC19质粒检查测量变为错误率达到108cfu/μg。 操 作 说 明1.取100μl雪上融入的L21(DE3)pLysS感悟态上皮上皮体細胞系，填加需求质粒并慢慢的混匀，雪上静置 2两半小时。2.42℃水浴热激45秒，较快放回雪上并静置2半小时，颤动会变低变为错误率。3.向离心力式管上填加700μl包涵抗菌素的没有细菌陪养基（2YT或LB），混匀后37℃，200rpm恢复60半小时4.5000rpm离心力式一半小时收菌，留取100μl以內上清慢慢的吹打重悬菌块并涂抹到含34 µg/ml氯霉素及合理质粒筛分抗菌素的2YT 或LB陪养基上。5.将板式反过来放于37℃陪养箱住宿陪养。注 意 事 项1.感悟态上皮上皮体細胞系最合适在雪上融入。2.渗入质粒时应当柔和操控。3.变为高渗透压的质粒可合理削减不可能用到涂板的菌量。4.成脂时，IPTG渗透压待选（0.1-2mM均可）5.为拥有要求量的蛋清，合适成脂時间，摄氏度，IPTG渗透压需实验性者优化提升。6.BL21(DE3)pLysS 菌株带入 pLysS质粒，除恢复陪养基为无抗菌素外，其他的书采用陪养基、陪养液均应包含34 µg/ml氯霉素，为了防止质粒丢弃。Sample Induction Protocol (for reference only)1.Inoculate a single colony from a freshly streaked plate into 5 ml 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 0.5-0.8.5.(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 3-4 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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