Tuner(DE3)pLysS Chemically Competent Cell
● 基因型
F- ompT hsdSB(rBmB- ) gal dcm lacY1 (DE3)pLysS CamR
● 产品说明
Tuner(DE3)菌株是 BL21 菌株的 lacZY 基因 (半乳糖苷透性酶基因)突变株,此突变导致 IPTG 以均一速度进入体系
中大肠杆菌的每个细胞,产生更加严格、均一的浓度依赖。将具有氯霉素抗性的 pLysS 质粒导入 Tuner(DE3)细胞
中即是 Tuner(DE3) pLysS,pLysS 表达 T7 溶菌酶,T7 溶菌酶可以与 T7 RNA 聚合酶结合抑制其转录活性,进而
降低目的基因的背景表达水平,但不干扰 IPTG 诱导的表达,非常适合毒性蛋白的原核表达。该菌株染色体整合了
λ 噬菌体 DE3 区 (DE3 区含有 T7 噬菌体 RNA 聚合酶),可同时表达 T7 RNA 聚合酶和大肠杆菌 RNA 聚合酶,可
用于 pET 系列,pGEX,pMAL 等质粒的蛋白表达。Tuner(DE3)pLysS 感受态细胞由特殊工艺制作,pUC19 质粒检
测转化效率达 107 cfu/μg DNA。
● 操作方法
1. Tuner(DE3) pLysS 感受态细胞从-80℃拿出,迅速插入冰中,5 分钟后待菌块融化,加入目的质粒并用手拨打 EP
管底轻轻混匀(避免用枪吸打),冰中静置 25 分钟。
2. 42℃水浴热激 45 秒,迅速放回冰中并静置 2 分钟,晃动会降低转化效率。
3. 向离心管中加入 700 μl 不含抗生素的 LB 无菌培养液,37℃,200 rpm 复苏 60 分钟。
4. 5000 rpm 离心一分钟收菌,留取 50 μl 左右上清轻轻吹打重悬菌块并涂布到含相应抗生素的 LB 培养基上。
5. 将平板倒置放于 37℃培养箱过夜培养。
● 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).
IPTG 配制:
Prepare 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.
氯霉素配制
Chloramphenicol 34 mg/ml in ethanol. Store at -20℃. Use at 34 µg/ml.
● 注意事项
1. 感受态细胞最好在冰中缓慢融化,插入冰中 8 分钟内加入目标 DNA,不可在冰中放置时间过长,长时间存放
会降低转化效率。
2. 转化高浓度的质粒可相应减少最终用于涂板的菌量。
3. 诱导时,IPTG 浓度可选(0.1-10 mM 均可)。
4. 为获得需要量的蛋白,最佳诱导时间,温度,IPTG 浓度需实验者优化。
5. Tuner(DE3) pLysS 菌株携带 pLysS 质粒,除复苏培养基为无抗生素外,其余所用培养基、培养液均应含有
34 µg/ml 氯霉素,以防质粒丢失。
本产品仅供科研使用.请勿用于医药,不能用于临床治疗诊断使用!