A simple and rapid preparation of M13 sequencing templates for manual and automated dideoxy sequencing.
A simple and rapid preparation of M13 sequencing templates for manual and automated dideoxy sequencing.
复制标题
简单快速地制备 M13 测序模板，用于手动和自动双脱氧测序。
作者：
T. Kristensen;H. Voss;W. Ansorge
本文介绍了一种制备M13单链DNA测序模板的简便、快速的方法。用乙酸从培养基上清液中沉淀噬菌体，并在玻璃纤维过滤器上回收。随后，在DNA结合到玻璃上的同时，进行DNA的解离和污染物的去除。最后，将纯化的DNA在小体积的低盐缓冲液中洗脱。产率高于标准方法。简化的程序需要不到30分钟，不需要特殊的技能或设备;序列分辨率与通过标准程序获得的Klenow片段和T7 DNA聚合酶，放射性标记以及荧光标记的自动测序一样好。
A simple and rapid procedure for the preparation of M13 single stranded DNA sequencing templates which does not involve phenol extractions and alcohol precipitations is described. Bacteriophages are precipitated from media supernatants with acetic acid and recovered on glass fiber filters. Subsequent dissociation of the phages and removal of contaminants is performed while the DNA is bound to the glass. Finally, the purified DNA is eluted in a small volume of low-salt buffer. The yield is higher than that obtained by standard methods. The simplified procedure takes less than 30 minutes and does not demand special skills or equipment; the sequence resolution is as good as that obtained by standard procedures both with the Klenow fragment and T7 DNA polymerase, with radioactive labelling as well as in automated sequencing with a fluorescent label.