Tuner(DE3)pLysS

Tuner(DE3)pLysS Chemically Competent Cell 產品說明書

產品規格 （CAT#: EC1051）

Tuner(DE3)pLysS：                                     100μl/支

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

保存條件(保質期)：                             -80℃（6個月） 

基因型

F^- ompT hsdS_B(r_B^- m_B^- ) gal dcm lacY1 (DE3)pLysS Cam^R

產品說明

Tuner(DE3)菌株是BL21菌株的lacZY基因 (半乳糖苷透性酶基因)突變株，此突變導致IPTG以均一速度進入體系中大腸桿菌的每個細胞，產生更加嚴格、均一的濃度依賴。將具有氯霉素抗性的pLysS質粒導入Tuner(DE3)細胞中即是Tuner(DE3) pLysS，pLysS表達T7溶菌酶，T7溶菌酶可以與T7 RNA聚合酶結合抑制其轉錄活性，進而降低目的基因的背景表達水平，但不干擾IPTG誘導的表達，非常適合毒性蛋白的原核表達。該菌株染色體整合了λ噬菌體DE3區 (DE3區含有T7噬菌體RNA聚合酶)，可同時表達T7 RNA聚合酶和大腸桿菌RNA聚合酶，可用于pET系列，pGEX，pMAL等質粒的蛋白表達。Tuner(DE3)pLysS感受態細胞由特殊工藝制作，pUC19質粒檢測轉化效率達10⁷ cfu/μg DNA。

操作方法

1. Tuner(DE3) pLysS感受態細胞從-80℃拿出，迅速插入冰中，5分鐘后待菌塊融化，加入目的質粒并用手撥打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，迅速放回冰中并靜置2分鐘，晃動會降低轉化效率。

3. 向離心管中加入700 μl不含抗生素的LB無菌培養液，37℃，200 rpm復蘇60分鐘。

4. 5000 rpm離心一分鐘收菌，留取50 μl左右上清輕輕吹打重懸菌塊并涂布到含相應抗生素的LB培養基上。

5. 將平板倒置放于37℃培養箱過夜培養。

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.

注意事項

1. 感受態細胞最好在冰中緩慢融化，插入冰中8分鐘內加入目標DNA，不可在冰中放置時間過長，長時間存放會降低轉化效率。

2. 轉化高濃度的質粒可相應減少最終用于涂板的菌量。

3. 誘導時，IPTG濃度可選（0.1-10 mM均可）。

4. 為獲得需要量的蛋白，最佳誘導時間，溫度，IPTG濃度需實驗者優化。

5. Tuner(DE3) pLysS 菌株攜帶 pLysS質粒，除復蘇培養基為無抗生素外，其余所用培養基、培養液均應含有34 μg/ml氯霉素，以防質粒丟失。

說明書下載

Tuner(DE3)pLysS Chemically Competent Cell 產品說明書