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C43(DE3) pLysS

文字:[大][中][小] 2018-1-2    浏览次数:869    

C43(DE3) pLysS Chemically Competent Cell 产品说明书




产品规格 (CAT#: EC1041)

C43(DE3) pLysS:                                       100μl/支

pUC19 (control vector,10pg/μl):                    10μl

保存条件(保质期):                             -80℃(6个月) 


基因型

FompT hsdSB (rB- mB-) gal dcm (DE3)pLysS CamR


产品说明

OverExpress C43(DE3)、OverExpress C41(DE3)两个菌株均起源于BL21(DE3),其优点是可以高效表达毒性蛋白或疏水性蛋白。OverExpress C41(DE3)跟BL21(DE3)的区别在于其基因组含有至少一个未知突变,这个未知突变使其获得了高效表达毒性蛋白的能力,此突变位点参与大肠杆菌表达毒性蛋白时的细胞死亡途径;OverExpress C43(DE3)来源于OverExpress C41(DE3),是通过筛选OverExpress C41(DE3)对另一个不同毒性蛋白的抗性菌株获得。C43(DE3)菌株具有比C41(DE3)更强的表达毒性蛋白和疏水性蛋白的能力。将具有氯霉素抗性的pLysS质粒导入C43(DE3)细胞中即是C43(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。C43(DE3)pLysS感受态细胞由特殊工艺制作,pUC19质粒检测转化效率可达1×108 cfu/μg DNA。


操作方法

1. C43(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34 µg/ml氯霉素及所选质粒筛选抗生素的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(0.1-2 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. C43(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。





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C43(DE3) pLysS Chemically Competent Cell 产品说明书

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