ProClone™ 高效能Competent cell勝任細胞 | ABMGOOD貨號E003

具備有高效能的Competent cell勝任細胞，是cloning技術基本要領。ProClone™讓Competent cell揚名全世界。其高效能的能力，讓cloning更容易，在一分鐘就做完Transformation。轉化效率：>1 x 10^6 CFU/μg DNA。產生高質量的質粒製劑 (endA1)、抗 T1 噬菌體 (fhuA2)、重組率降低 (recA1)、藍/白篩選能力。

Features:

Transformation efficiency: >1 x 10^6 CFU/µg of DNA
Results in high quality plasmid preparations (endA1)
Resistant to T1 phage (fhuA2)
Exhibits reduced recombination rate (recA1)
Blue/White screening capable

Cat. No.	E003
Name	ProClone™ Competent Cells
Unit	5ml (4 x 1.25ml)
Category	Molecular Biology Enzymes and Kits
Description	ProClone™ Competent Cells are high-efficiency, chemically competent DH5α (E. coli) cells, ideal for both routine and challenging plasmid amplification and subcloning projects.
Expression System General	E. coli
Storage Condition	Store at -80°C.

Protocol

The following protocol serves as a general guideline and may require optimization.

1. Thaw 1 tube of cells on ice for 10-15 minutes, then gently invert tube to thoroughly mix any settled cells. Aliquot 60 µl of cells into a sterile 1.5 ml tube per transformation. Remaining cells can be aliquoted and re-frozen for later use, but expect lower performance from re-frozen cells.

2. Add 10 pg-100 ng of plasmid DNA or 5-10 µl of the subcloning reaction (i.e. ligation) to the cells and mix gently. Incubate the mixture at 4°C for 30 minutes.

3. Heat shock the cells using a waterbath or heat block at 42°C for 45 seconds, then immediately place on ice for 1-2 minutes.

4. Add 150 µl of sterile LB broth containing no antibiotics and incubate the cells at 37°C in a shaking incubator for 1 hour for recovery.

5. Plate the entire volume on LB agar containing appropriate antibiotics and incubate overnight at 37°C.

【Competent cell勝任細胞注意事項】

1.本建議濃度以新鮮配製之抗生素為準。

2.使用過期之抗生素，將因藥性不足，易產生假性轉形菌落。

3.若使用過量的抗生素濃度或同時使用多種抗生素進行篩選，轉形效率會明顯下降。請務必依建議濃度進行抗生素篩選

【藍/白篩選】

進行轉形前，請先確認質體帶有LacZ operon。轉形後，請將勝任細胞塗佈於含0.5 mM IPTG 和 40~60 μg/ml X-gal之LB培養基。37^oC培養後，白色菌落表示外源DNA嵌入LacZ operon，藍色菌落則仍具備正常的β-galactosidase代謝能力。

【含抗生素之LB培養基配方】

10 g/L Tryptone 5 g/L Yeast extract 10 g/L NaCl 15 g/L Agar 以NaOH調整酸鹼值至pH7.5，高溫高壓滅菌後，待降溫至55oC，加入適當濃度之抗生素。

LB Agar (Miller配方)培養基 | 貨號NCM0142A

LB培養基（Lysogeny broth，LB）廣泛解釋爲Luria-Bertani培養基，依據其發明人貝爾塔尼（Giuseppe Bertani） 的說法，這個名字來源於英語的 Lysogeny broth，即溶菌培養基。Luria-Bertani培養基是微生物學實驗中最常用的營養性培養基，用於培養大腸桿菌(E. coli i) 等細菌，Broth加入瓊脂(Agar)製成的固態培養基。加入抗生素的 LB 培養基(LB Broth) 可用於篩選大腸桿菌爲宿主的cloning。

LB Agar（Miller）是一種營養豐富的培養基，設計用於培養重組菌(recombinant strains) 的純培養物，它基於Miller描述的配方。大腸桿菌在LB培養基中生長至對數晚期 (late log phase)。 一些plasmid vectors複製到high copy number並且不需要選擇性擴增。 一些載體不能如此自由地複制並且需要被選擇性地擴增。 可加入氯黴素(Chloramphenicol)以抑制宿主合成(host synthesis)並防止細菌染色體(bacterial chromosome)的複製。LB Agar，Miller含有10 g / L的氯化鈉，與Lennox配方中的含量不同。 這允許研究人員為特定菌株選擇最佳鹽濃度。 培養基可以無菌補充葡萄糖glucose或抗生素antibiotics。註明: Product was previously known as Acumedia Product: 7213 LB Agar (Miller); and LabM Product: LAB168 LB Agar (Miller)。氯化鈉的量經常隨細菌的生長需求不同而有所改變LB-Miller (10 g/L NaCl)、LB-Lennox (5 g/L NaCl)、LB-Luria (0.5 g/L NaCl)

胰蛋白腖 (Tryptone )————————————- 10.0 g / L

酵母抽提物(Yeast Extract ) ———-5.0g / L

Sodium Chloride ————————10.0g / L

Agar _________________15.0 g/L