保存條件： -80℃

基 因 型

F– ompT hsdS(rB– mB–) dcm+ galλ(DE3) endA Hte [argUileY leuW Camr ]

簡 要 說 明

AngYuBio BL21-CodonPlus(DE3)-RIL菌株來源于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) 對應(yīng)的tRNA （argU、ileY、leuW），提高外源基因，尤其是富含AT-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)，具有氯霉素抗性。 BL21- Codon Plus(DE3)-RIL 感受態(tài)細(xì)胞由特殊工藝制作，經(jīng)pUC19質(zhì)粒檢測轉(zhuǎn)化效率高達(dá)108cfu/μg。

操 作 說 明

1.取100μl冰上融化的AngYuBio BL21-CodonPlus(DE3)-RIL感受態(tài)細(xì)胞，加入目的質(zhì)粒并輕輕混勻，冰上靜置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2YT或LB培養(yǎng)基，混勻后37℃，200rpm復(fù)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相應(yīng)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)。

注 意 事 項(xiàng)

1.AngYuBio 感受態(tài)細(xì)胞最好在冰上融化。

2.混入質(zhì)粒時應(yīng)輕柔操作。

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

4.誘導(dǎo)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(dǎo)時間，溫度，IPTG濃度需實(shí)驗(yàn)者優(yōu)化。

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 untilneeded 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 x g for 10minutes 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-Dthiogalactoside; 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.