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如何处理 收到的克隆 BAC/ PAC

2021-04-25

The clones you receive from us are usually shipped in LB agar stab culture format. Most (but not all) of the BAC or PAC clones are propagated in the DH10B E.coli host (either the original strain DH10B or the later T1-phage resistant version of DH10B). The bacteria harboring the plasmid (BAC, PAC, fosmid, cDNA or vector) have been inocculated into LB agar (containing 12.5ug/ml chloramphenicol for BAC clones and 25 ug/ml kanamycin for PAC clones, or other antibiotics as appropriate for the specific clone). The LB agar is placed in a small tube and this arrangement is called a "stab-culture" by microbiologists. Stab cultures are typically shipped at ambient ("room") temperature and should be placed in a refridgerator (40C) upon arrival. The few days at room temperature don't harm the plasmid. However, please note that the culture has a finite life at 4°C.

We recommend that you streak this culture to single colonies on a LB agar plate with the appropriate antibiotic and prepare a glycerol stock of the clones for long term storage at -85°C. Please make sure also to confirm the content of the clones by your own favorite method (for instance PCR for a sequence expected to be unique for the clone). While our libraries have minimal well to well contamination, some colonies may not contain the expected plasmids. In most cases this is due to a database mismatch (see our Q&A). Hence, it is important, before initiating any experiments, to check 3-5 colonies (PCR, or diagnostic restriction digest). This to make sure that the clone stock that we sent you is pure and represents the gene or sequence of interest.

How would you be able to know which  E.coli host, cloning vector and antibiotic resitance apply to your clone?  That is typically described on a webpage for the clone collection ("library") which contains the specific clone. For instance, the clone "RP11-163M23" is a member of the RP11 library (full name "RPCI-11"). In the left top corner of the current webpage, you will see a Google Site-Search Box). Type "RP11'" or "RPCI-11" to find other pages on our site with details related to this library. One of these pages contains a detailed description of the library and literature references: https://bacpacresources.org/hmale11.htm. Dish "163" contains 384 different BAC clones created in the vector pBACe3.6 and host DH10B. The requested clone is found at the intersection of row "M" and column "23". The vector defines the antibiotic resistance gene (nearly always chloramphenicol).

您从我们这里收到的克隆通常以LB琼脂刺培养形式运输。大多数(但不是全部)BAC或PAC克隆在DH10B 大肠杆菌宿主(原始菌株DH10B或后来的DH10B T1噬菌体抗性版本)中繁殖。携带质粒的细菌(BAC,PAC,粘粒,cDNA或载体)已接种到LB琼脂(对于BAC克隆,含有12.5ug / ml氯霉素,对于PAC克隆,含有25 ug / ml卡那霉素),或其他针对具体情况的抗生素克隆)。将LB琼脂放在一个小管中,这种方法被微生物学家称为“稳定培养”。这种形式通常在室温(“房间”0 C)运输到达。在室温下几天不会损害质粒。但是,请注意,这种x形式在4°C下具有有限的寿命。

我们建议您将这种培养物在带有适当抗生素的LB琼脂平板上划线成单个菌落,并准备该克隆的甘油储备,以在-85°C下长期保存。还请确保通过您自己喜欢的方法来确认克隆的内容(例如,PCR预期该克隆唯一的序列)。虽然我们的文库对各孔的污染极小,但某些菌落可能不包含预期的质粒。在大多数情况下,这是由于数据库不匹配(请参见我们的问答))。因此,在开始任何实验之前,重要的是要检查3-5个菌落(PCR或诊断性限制性消化)。这可以确保我们发送给您的克隆种群是纯净的,并且代表目的基因或序列。

您将如何知道哪个大肠杆菌宿主,克隆载体和抗生素耐药性适用于您的克隆?  通常在包含特定克隆的克隆集合(“库”)的网页上进行描述。例如,克隆“ RP11-163M23”是RP11库的成员(全名“ RPCI-11”)。在当前网页的左上角,您会看到一个Google Site-Search Box。键入“ RP11'”或“ RPCI-11”以在我们网站上找到其他与此库有关的页面。这些页面之一包含对库的详细描述和参考文献:https : //bacpacresources.org/hmale11.htm。皿“ 163”包含在载体pBACe3.6和宿主DH10B中创建的384个不同的BAC克隆。在行“ M”和列“ 23”的相交处找到了所请求的克隆。该载体定义了抗生素抗性基因(几乎总是氯霉素)。



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