| 简易快速高效制备T载体 |
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| 引用本文： | 徐锋,武晓丽.简易快速高效制备T载体[J].安徽农业科学,2008,36(2):460-461. |
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| 作者姓名： | 徐锋 武晓丽 |
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| 作者单位： | 1. 长江大学生命科学学院,湖北荆州,434025
2. 中国农业科学院植物保护研究所,北京,100094 |
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| 摘 要： | 目的]制备成本廉价、使用方便、效率高的T载体。方法]用PstI充分酶切纯化的质粒pUC19,再用T4DNA聚合酶消化15min,电泳得到T载体,进行质量检测,并与商品化的T载体pMD18-T进行对比。结果]自制的T载体自连检测只发现2个菌落与PCR扩增片段连接。经转化大肠杆菌JM109,pMD18-T的平均白斑率为99.4%,转化菌落为3.55万个/μg,自制T载体的平均白斑率为99.7%,转化菌落为3.38万个/μg,完全可以与商品出售的T载体媲美。结论]该研究自制的T载体自连率低,假阳性率低,克隆效率高,可替代商品化的T载体用于TA连接,而且制备时间短,成本低廉,技术简单,值得推广。
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| 关 键 词： | T载体 PstI PCR 克隆载体 |
| 文章编号： | 0517-6611（2008）02-00460-02 |
| 收稿时间： | 2007-09-05 |
| 修稿时间： | 2007年9月5日 |
A Simple, Rapid and High Efficient Method for Preparing T-vector |
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| XU Feng et al.A Simple, Rapid and High Efficient Method for Preparing T-vector[J].Journal of Anhui Agricultural Sciences,2008,36(2):460-461. |
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| Authors: | XU Feng |
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| Abstract: | Objective] The study aimed to prepare T-vector with low cost, convenience in use and high efficiency. Method] T-vector was obtained through doing sufficient enzymolysis with Pst I on purified plasmid pUC19, then digesting with T4 DNA polymerase for 15 min and finally carrying out electrophoresis, which was conducted for quality detection and compared with commercialized T-vector pMD18-T. Result] Only 2 colonies connected with PCR amplified fragments were found in self-connection detection of self-made T-vector. The average white spot rate of translated Escherichia coli JM109 and pMD18-T was 99.4 % with translated colonies being 3.55×104 /μg. The average white spot rate of self-made T-vector was 99.7 % with translated colonies being 3.38×104 /μg, which could be compared with T-vector sold as commodity completely. Conclusion] The self-made T-vector in the research had low self-connection rate, low false-positive rate and high clone efficiency, and could replace commercialized T-vector to use in TA connection with short preparation time, low cost and simple technology, was worthy of popularizing. |
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| Keywords: | T-vector Pst I PCR Cloning vector |
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