BL21(DE3) Chemically Competent Cell 產品說明書
產品規格 （CAT#: EC1002）
BL21(DE3)： ; 100μl/支
pUC19 (control vector，10pg/μl): 10μl
保(bao)存條(tiao)件(保(bao)質期)： -80℃（6個月）
基因(yin)型(xing)
F- ompT hsdSB(rB- mB- ) gal dcm(DE3)
產品說明
BL21(DE3)菌株用于高效表達克隆于含有噬菌體T7啟動子的表達載體(如pET系列)的基因。λ噬菌體DE3區含有T7噬菌體RNA聚合酶，該區整合于BL21的染色體上，所以稱為BL21(DE3)。可同時表達T7 RNA聚合酶和大腸桿菌RNA聚合酶，用于pET系列，pGEX，pMAL等質粒的蛋白表達。BL21(DE3)感受態細胞由特殊工藝制作，pUC19質粒檢測轉化效率達107cfu/μg DNA。
操作(zuo)方法
1.BL21(DE3)感受態細(xi)胞從-80℃拿出，迅(xun)速插入冰中(zhong)，5分鐘后待菌塊融化，加入目(mu)的DNA（質粒或連接(jie)產物）并用手撥(bo)打EP管底輕(qing)(qing)輕(qing)(qing)混勻(yun)(避(bi)免用槍吸打)，冰中(zhong)靜(jing)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(dong)會降低轉化(hua)效率。
3.向離心管中加入700μl不(bu)含抗生素的(de)無菌培養基(2YT或LB)，混(hun)勻(yun)后(hou)37℃，200rpm復蘇60分鐘。
4.5000rpm離心一分鐘(zhong)收(shou)菌，留取(qu)100μl左(zuo)右上清輕輕吹打重懸菌塊并(bing)涂布(bu)到含相應抗生素的2YT或LB培養(yang)基上。
5.將平(ping)板倒置放(fang)于(yu)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 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) bydissolving 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. 感受態細(xi)胞最好在冰中(zhong)緩(huan)慢融化，插入冰中(zhong)8分鐘內加入目標DNA，不可在冰中(zhong)放(fang)置(zhi)時間過長，長時間存放(fang)會降低(di)轉化效率(lv)。
2. 混(hun)入質(zhi)粒(li)時應輕柔(rou)操作(zuo)。
3. 轉化高濃(nong)度的質粒可(ke)相應減少最終用于涂(tu)板的菌量。
4. 誘導時，IPTG濃度可選（0.1-2mM均可）。
5. 為獲得(de)需要量的(de)蛋白，最佳誘導時間，溫度，IPTG濃(nong)度需實驗(yan)者優化。
