pUC18 DNA - FAQs

无克隆产生的原因可能是出现了下列问题：

•细菌不是感受态细胞。使用包含在One Shot模块内的pUC18载体检查感受态细胞的转化效率。
•平板的抗生素浓度不正确，或者平板过于陈旧。使用100 μg/mL的氨苄青霉素或50 µg/mL的卡那霉素。确保氨苄平板是新鲜的（储存时间小于1个月）。
•产物被磷酸化了（仅针对TOPO克隆而言）。磷酸化的产物可以进行TA-克隆但不能进行TOPO-克隆。这是因为连接所需的磷酸基团已经包含在载体DNA和拓扑异构酶-形成的中间体复合物中了。TOPO载体含有一个与拓扑异构酶共价结合的3' 磷酸基团和一个5'磷酸基团。非TOPO载体（TA和Blunt）含有一个3' OH基团和一个5'磷酸基团。磷酸化产物在进行TOPO-克隆之前应该用磷酸酶（CIP）处理。

No colonies may occur due to the following problems:

Bacteria were not competent. Use the pUC18 vector included with the One Shot module to check the transformation efficiency of the cells.
- Incorrect concentration of antibiotic on plates, or the plates are too old. Use 100 µg/mL of ampicillin or 50 µg/mL kanamycin. Be sure ampicillin plates are fresh (< 1 month old).
- The product was phosphorylated (TOPO cloning only). Phosphorylated products can be TA-cloned but not TOPO-cloned. This is because the necessary phosphate group is contained within the topoisomerase-DNA intermediate complex of the vector. The TOPO vector has a 3' phosphate to which topoisomerase is covalently bound and a 5' phosphate. The non- TOPO vectors (TA and Blunt) have a 3' OH and a 5' phosphate. Phosphorylated products should be phosphatased (CIP) before TOPO-cloning.