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BL21 Star(DE3) Chemically Competent Cell 產(chǎn)品說明書 

產(chǎn)品規(guī)格

BL21 Star(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 rne131 (DE3)

產(chǎn)品說明

BL21 Star(DE3)菌株源于BL21(DE3)菌株，含有rne131突變（RNaseE基因），RNaseE基因的突變降低了內(nèi)源RNase的積累，增強菌株細(xì)胞內(nèi)mRNA的穩(wěn)定性，從而提高異源蛋白的表達(dá)水平。主要適用于T7啟動子表達(dá)載體(如pET系列)的高水平蛋白表達(dá)，同時含有大腸桿菌RNA聚合酶，也可用于非T7啟動子表達(dá)載體（pGEX，pMAL等）的蛋白表達(dá)。由于BL21 Star(DE3)菌株的異源基因基礎(chǔ)表達(dá)水平較高，所以不適合毒性蛋白的表達(dá)。BL21 Star(DE3)感受態(tài)細(xì)胞由特殊工藝制作，pUC19質(zhì)粒檢測轉(zhuǎn)化效率達(dá)10⁸cfu/μg DNA。

操作方法

1.  BL21 Star(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，晃動會降低轉(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℃，200rpm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 min 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

1. 感受態(tài)細(xì)胞最好在冰中緩慢融化，插入冰中8分鐘內(nèi)加入目標(biāo)DNA，不可在冰中放置時間過長，長時間存放會降低轉(zhuǎn)化效率。

2. 混入質(zhì)粒時應(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，IPTG濃度可選（0.1-2mM均可）。