新聞動態

M15(pREP4)蛋白表達菌株含有pREP4質粒，該質粒通過p15A復制子啟動復制，可以與許多原核表達質粒共存于同一大腸桿菌細胞中；同時該質粒攜帶lac I基因，可高效表達lac 抑制蛋白，在IPTG誘導前可抑制目標蛋白的本底表達，特別適合毒性基因的表達。M15(pREP4)菌株是PQE系列質粒的配套菌株，特別適合PQE系列質粒或由E.coli T5 啟動子誘導表達的質粒的蛋白表達。pREP4質粒賦予該菌株卡那霉素抗性。唯地生物生產的M15(pREP4)感受態細胞經特殊工藝制作，pUC19質粒檢測轉化效率達2×10⁸cfu/μg DNA。

操作方法

1. M15(pREP4)感受態細胞從-80℃拿出，迅速插入冰中，5分鐘后待菌塊融化，加入目的質粒并用手撥打EP管底混勻，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，晃動會降低轉化效率。

3. 向離心管中加入700 μl不含抗生素的無菌培養基 (LB)，混勻后37℃，200 rpm復蘇60分鐘。

4. 5000 rpm離心一分鐘收菌，留取50 μl左右上清輕輕吹打重懸菌塊并涂布到含相應抗生素的LB培養基上（若質粒濃度較高，也可稀釋后涂板，務必保證能在平板上挑到單克隆菌落）。

5. 將平板倒置放于37℃培養箱過夜培養，M15(pREP4)菌株在平板或液體培養基中生長時，需在培養基中加入終濃度為35ug/ml的卡那霉素。

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 accep- table).

IPTG配制：

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. 感受態細胞最好在冰中緩慢融化，插入冰中8分鐘內加入目標DNA，不可在冰中放置時間過長，長時間存放會降低轉化效率。

2. M15(pREP4)菌株在平板或液體培養基中生長時，需在培養基中加入終濃度為35ug/ml的卡那霉素。

3. 轉化高濃度的質粒可相應減少最終用于涂板的菌量。

4. 為獲得需要量的蛋白，最佳誘導時間，溫度，IPTG濃度需實驗者優化。

說明書下載

M15(pREP4) Chemically Competent Cell 產品說明書

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