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采用Trizol试剂提取副溶血弧菌感染的九孔鲍血细胞的总RNA,经Oligotex纯化得到mRNA.根据SMART技术原理合成双链cDNA.双链cDNA采用双链特异核酸酶进行cDNA的均一化,构建成九孔鲍血细胞的均一化全长cDNA文库.原始文库的库容为3.4×106cfu/cm3,重组率为92.3%,扩增后文库的滴度为2.6×1011cfu/cm3以上.从文库中随机挑取897个克隆,测序获得高质量ES-Ts814条,其中有23条Contigs;762条Singlets,Unigenes共785条,冗余率只有3.56%.以上结果说明所构建的文库质量好,完全可以满足后续基因克隆和表达序列标签测序工作的需要.
Trizol reagent was used to extract the total RNA of hemolymph of V. parahaemolyticus and the mRNA was purified by Oligotex.The double-stranded cDNA was synthesized according to the principle of SMART technique.The double-stranded cDNA was double-stranded cDNA The library was a uniform full-length cDNA library of nine-hole abalone blood cells.The library had a capacity of 3.4 × 106 cfu / cm3, a recombination rate of 92.3%, and a titer of 2.6 × 1011 cfu / cm3 after amplification.The library was randomly selected 897 clones were sequenced to obtain high quality ES-Ts814, of which 23 Contigs, 762 Singlets and 7 Unigenes with a redundancy rate of 3.56%. The above results show that the constructed library is of good quality and can meet the needs of the following genes The need to clone and express sequence tags for sequencing work.