BL21-CodonPlus(DE3)-RIPL Chemically Competent Cell 使用說(shuō)明書(shū)
產(chǎn)品說(shuō)明：
BL21-CodonPlus(DE3)-RIPL菌株來(lái)源于Stratagene公司的BL21-Gold菌株，缺少Lon蛋白酶和OmpT蛋白酶，從而減少對(duì)重組蛋白的降解，補(bǔ)充大腸桿菌缺乏的4種稀有密碼子 (AGA, AUA, CCC, CUA)對(duì)應(yīng)的tRNA (argU, ileY, proL, leuW)，提高外源基因，尤其是富含AT-或 GC-的真核基因在原核系統(tǒng)中的表達(dá)水平。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)，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)，同時(shí)具有四環(huán)素，氯霉素，鏈霉素，壯觀霉素抗性。BL21- CodonPlus(DE3)-RIPL 感受態(tài)細(xì)胞由特殊工藝制作，pUC19質(zhì)粒檢測(cè)轉(zhuǎn)化效率高達(dá)108 cfu/µg。
產(chǎn)品組成：
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組 分
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BL21-CodonPlus(DE3)-RIPL Chemically Competent Cell
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10 × 100 µL
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50× 100 µL
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pUC19（control vector，10pg/μL）
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10μL
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10μL
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* 基因型：F–ompT hsdS(rB–mB–) dcm+ TetR gal λ(DE3) endA Hte [argU proL CamR] [argU ileY leuW Strep/SpecR]
儲(chǔ)存條件：
-80℃±10℃保存6個(gè)月。請(qǐng)勿將本品置于-20℃或液氮中保存。
使用方法：
1. 將感受態(tài)細(xì)胞從-80℃冰箱中取出，置于冰水浴中融化。
2. 將目的DNA（質(zhì)粒或連接產(chǎn)物）加入到100μL感受態(tài)細(xì)胞中，輕輕彈勻，冰上孵育25分鐘。
3. 42℃水浴熱激45秒，立刻置于冰上，靜置2～3分鐘，晃動(dòng)會(huì)降低轉(zhuǎn)化效率。
4. 向離心管中加入200μL無(wú)抗LB、TB或SOC培養(yǎng)基，混勻后于220rpm，37℃搖床振蕩復(fù)蘇60分鐘，使轉(zhuǎn)化物表達(dá)抗性基因。
5. 根據(jù)實(shí)驗(yàn)要求吸取適量菌液，涂布至相應(yīng)抗生素的2YT或LB培養(yǎng)基上，室溫正置10分鐘，待菌液被平板充分吸收后，37℃倒置過(guò)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 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)胞冰水浴中緩慢融化。插入冰中8分鐘內(nèi)加入目的DNA，不可在冰上放置時(shí)間過(guò)長(zhǎng)，長(zhǎng)時(shí)間存放會(huì)降低轉(zhuǎn)化效率。
2. 待轉(zhuǎn)化DNA加入體積不要超過(guò)感受態(tài)細(xì)胞體積的1/10。
3. 待轉(zhuǎn)化DNA加入感受態(tài)細(xì)胞后，請(qǐng)勿用移液槍吹打，輕輕彈勻即可。
4. 為確保最高效率轉(zhuǎn)化，整個(gè)操作過(guò)程應(yīng)盡量輕柔，并保持冰上操作。
5. 轉(zhuǎn)化高濃度的質(zhì)粒或高效率的連接產(chǎn)物可相應(yīng)減少最終用于涂板的菌量。
6. 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。
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