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OverExpress C43(DE3) Chemically Competent Cell 產(chǎn)品說明書

產(chǎn)品規(guī)格

OverExpress C43(DE3):                     10×100μl

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

保存條件：-80℃

基因型

F^–ompT hsdS_B (r_B^- m_B^-) gal dcm (DE3)

產(chǎn)品說明

OverExpress C43(DE3)、OverExpress C41(DE3)兩個(gè)菌株均起源于BL21(DE3)，其優(yōu)點(diǎn)是可以高效表達(dá)毒性蛋白或疏水性蛋白。OverExpress C41(DE3)跟BL21(DE3)的區(qū)別在于其基因組含有至少一個(gè)未知突變，這個(gè)未知突變使其獲得了高效表達(dá)毒性蛋白 (1-5)的能力，此突變位點(diǎn)參與大腸桿菌表達(dá)毒性蛋白時(shí)的細(xì)胞死亡途徑；OverExpress C43(DE3)來源于OverExpress C41(DE3)，是通過篩選OverExpress C41(DE3)對另一個(gè)不同毒性蛋白的抗性菌株獲得。OverExpress C43(DE3)菌株具有比OverExpress C41(DE3)更強(qiáng)的表達(dá)毒性蛋白和疏水性蛋白的能力，所以說OverExpress C43(DE3) 菌株與BL21(DE3)相比擁有至少兩個(gè)未知突變，正是這兩個(gè)未知突變使其獲得了更廣泛的表達(dá)毒性蛋白或疏水性蛋白的能力。此菌株含有DE3區(qū)，可同時(shí)表達(dá)T7 RNA聚合酶和大腸桿菌RNA聚合酶，可用于pET系列，pGEX，pMAL等質(zhì)粒的蛋白表達(dá)。OverExpress C43(DE3)感受態(tài)細(xì)胞由特殊工藝制作，pUC19質(zhì)粒檢測轉(zhuǎn)化效率可達(dá)5×10⁸ cfu/μg DNA。

操作方法

1. OverExpress C43(DE3)感受態(tài)細(xì)胞從-80℃拿出，迅速插入冰中，5分鐘后待菌塊融化，加入目的DNA（質(zhì)?；蜻B接產(chǎn)物）并用手撥打EP管底輕輕混勻(避免用槍吸打)，冰中靜置25分鐘。

2. 42℃水浴熱激45秒，迅速放回冰上并靜置2分鐘，晃動(dòng)會(huì)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℃，200 rpm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)。

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×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

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.

注意事項(xiàng)

1. 感受態(tài)細(xì)胞最好在冰上融化。

2. 混入質(zhì)粒時(shí)應(yīng)輕柔操作。

3. 轉(zhuǎn)化高濃度的質(zhì)粒可相應(yīng)減少最終用于涂板的菌量。

4. 誘導(dǎo)時(shí)，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(shí)間，溫度，IPTG濃度需實(shí)驗(yàn)者優(yōu)化.