BL21 Chemically Competent Cell 產品說明書
產品規格 （CAT#: EC1001）
BL21： 100μl/支
pUC19 (control vector，10pg/μl): 10μl
保存條(tiao)件(保質期)： ; -80℃（6個(ge)月(yue)）
基因型
E. coli B F- dcm ompT hsdS(rB- mB-) gal [malB+]K-12(λS)
產品說明
BL21是最早開發的用于原核表達的菌株，BL21(DE3)、Rosetta、OrigamiB(DE3) 等一系列原核表達菌株均來源于BL21菌株。該菌株主要用于非毒性蛋白的表達，不含T7 RNA聚合酶，所以不能用于由T7啟動子驅動的蛋白表達(如：pET系列)；但含有大腸桿菌RNA聚合酶，可以用于tac或trc等使用大腸桿菌RNA聚合酶的原核系統的表達（如：pGEX，pMAL質粒）。BL21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。
操作方法
1. BL21感(gan)受態細胞從-80℃拿(na)出，迅速插入冰中，5分鐘后待菌塊融化，加入目的DNA（質粒或(huo)連接(jie)產物）并(bing)用手撥打EP管底輕輕混勻(yun)(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(hui)冰上(shang)并(bing)靜置(zhi)2分鐘，晃動(dong)會降低轉化效率。
3. 向離心管中加入(ru)700 μl不含抗(kang)生素的無菌培養(yang)基 (2YT或LB)，混勻后(hou)37℃，200 rpm復(fu)蘇60分鐘。
4. 5000 rpm離(li)心一分鐘(zhong)收菌，留(liu)取(qu)100 μl左右上(shang)清輕輕吹打重懸菌塊并(bing)涂(tu)布到含相應抗生素的2YT或LB培養基上。
5. 將(jiang)平板倒置放(fang)于37℃培養(yang)箱過夜(ye)培養(yang)。
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
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(gan)受態細胞最好在冰中(zhong)(zhong)緩慢融化，插入(ru)冰中(zhong)(zhong)8分(fen)鐘(zhong)內加入(ru)目標DNA，不可在冰中(zhong)(zhong)放置時間(jian)過長，長時間(jian)存(cun)放會(hui)降(jiang)低轉化效率。
2. 混入質粒時應輕柔操作。
3. 轉化高濃(nong)度(du)的(de)質粒可相(xiang)應減(jian)少最(zui)終用于涂板的(de)菌量。
4. 誘導時，IPTG濃度可選(xuan)（0.1-2 mM均可）。
5. 為獲得需要(yao)量的蛋白(bai)，最佳誘導時間，溫度，IPTG濃度需實(shi)驗者優(you)化。
