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Rosetta-gami(DE3)pLysS Chemically Competent Cell產(chǎn)品說明書

產(chǎn)品規(guī)格

Rosetta-gami(DE3)pLysS:                          10×100μl

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

保存條件：-80℃

基因型

Δ(ara-leu)7697ΔlacX74ΔphoA PvuII phoR araD139 ahpC galE galK rpsL(DE3) F′[lac⁺ lacI^q pro] gor522::Tn10trxB::kan pLysSRARE2 (Cam^r, Str^r, Tet^r)

產(chǎn)品說明

Rosetta-gami(DE3)pLysS 菌株聚合了不同原核表達菌株的優(yōu)勢：

＊ Rosetta-gami賦予其 Rosetta和Origami的優(yōu)點——補充大腸桿菌缺乏的6 種稀有密碼子(AUA, AGG, AGA, CUA, CCC, GGA)對應的tRNA，提高外源基因的表達水平，并且包含突變的硫氧還蛋白還原酶(thioredoxin reductase) (trxB)和谷胱甘肽還原酶(glutathione reductase) (gor)基因，它們是還原途徑的兩個關(guān)鍵酶，其突變有利于高效形成正確折疊的含有二硫鍵的蛋白，增強蛋白的可溶性。

＊ 該菌株染色體整合了λ噬菌體DE3區(qū) (DE3區(qū)含有T7噬菌體RNA聚合酶)適合T7啟動子誘導的蛋白表達。

＊ 該菌株攜帶的pLysS質(zhì)粒含有表達T7溶菌酶的基因，能夠降低目的基因的背景表達水平，但不干擾IPTG誘導的表達，適合表達毒性蛋白和非毒性蛋白。Rosetta-gami(DE3)pLysS 菌株具有卡那霉素，氯霉素，鏈霉素，四環(huán)素抗性，為亮氨酸生長缺陷型菌株。Rosetta-gami(DE3)pLysS感受態(tài)細胞由特殊工藝制作，pUC19質(zhì)粒檢測轉(zhuǎn)化效率高達10⁸ cfu/μg DNA。

操作方法

1. Rosetta-gami(DE3)pLysS感受態(tài)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34 μg/ml氯霉素及所選質(zhì)粒篩選抗生素的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.

氯霉素

Chloramphenicol  34 mg/ml in ethanol. Store at -20℃. Use at 34 μg/ml.

注意事項

1. 感受態(tài)細胞最好在冰中緩慢融化，插入冰中8分鐘內(nèi)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(zhuǎn)化效率。

2. 混入質(zhì)粒時應輕柔操作。

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

4. 誘導時，IPTG濃度可選(0.1-2mM均可)。

5. 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(yōu)化。

6. BL21(DE3)pLysS 菌株攜帶 pLysS質(zhì)粒，除復蘇培養(yǎng)基為無抗生素外，其余所用培養(yǎng)基、培養(yǎng)液均應含有34 μg/ml氯霉素，以防質(zhì)粒丟失。

7. 具有卡那霉素抗性，不能用于具有卡那霉素抗性質(zhì)粒的表達。