摘要
在随机六聚体引物浓度不变条件下 ,分别采用 0 .5、2、5μg草鱼呼肠孤病毒 (GCRV)单一片段RNA进行反转录cDNA合成及文库构建。结果表明 ,在GCRV RNA模板浓度大于 2 μg时 ,第二链cDNA合成产物可直接通过EB染色的琼脂糖凝胶电泳进行定量及鉴定。选择cDNA与载体连接比率为 5∶1,在高效电转化条件下 ,可获得 10 6 转化子 /μgcDNA的转化效率。阳性克隆子经酶切及PCR鉴定 ,约 4 5%以上的插入片段大于 50 0bp。
Under the condition of constant concentration of random hexamers, the methods of cDNA synthesis and library construction were described by using 0.5 μg, 2 μg and 5 μg individual RNA segment of GCRV as templates respectively. The result showed that when the template concentration of GCRV RNA was equal to or more than 2 μg, the products of synthesized cDNA could be analysed directly through agarose gel electrophoresis stained by EB. When the ligation ratio of cDNA and vector was 5∶1, cloning efficiency could be as high as 10 6 transformants gene rated per μg cDNA basing on highly efficient electroporation transformation. Up to 45% inserts of the segments were larger than 500 bp by restriction analysis and PCR amplification.
出处
《中国病毒学》
CSCD
2000年第1期78-82,共5页
Virologica Sinica
基金
国家自然科学基金!批准号 :395 70 0 35
