大规模筛选表达序列标签(ESTs)方法的改进

Improvement of the Large-scale Screening Method for Expressed Sequence Tags(ESTs)

介绍一种改进的大规模ESTs筛选的方法。以SMARTTM cDNA Library Construction Kit所构建的毛冠鹿大脑cDNA文库为研究材料,基于该文库噬菌体可自发转化为质粒的特点,直接把噬菌体文库转化为质粒文库。随机选取平板中转化后的质粒cDNA文库克隆,振荡培养后运用菌液电泳的方法代替传统的PCR筛选法,对重组克隆进行筛选。改进的菌液电泳法可以准确鉴定出重组的质粒,并估计出插入片段的大小。该方法能更好地保持SMARTTM cDNA Library Construction Kit所构建文库的完整性,是一种简单、高效、适用于大规模表达序列标签筛选的有效方法。

The aim of the research was to introduce an improved method for large-scale screening FiSTs. With cerebrum cDNA library of tufted deer con-structed by SMARTTM cDNA Library Construction Kit as research materials, based on characteristic of the library phage that could spontaneously translate into plasmid, the phage library was directly translated into plasmid library. Transformed plasmid cDNA library clones in flat were randomly selected. After shaking culture the bacterial liquid electrophoresis method was used to screen recombinant clones instead of traditional PCR screening method.Improved bacterial liquid electrophoresis method could identify recombinant plasmid exactly and evaluate the length of inserted fragments. This method could better keep the integrality of the library constructed by SMARTTM cDNA Library Construction Kit, so, it was a simple and efficient method which was suitable for large-scale ESTs screening.