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目的 构建金仓鼠神经管cDNA文库,以筛选与神经管发育及神经管畸形发生相关的基因。 方法 用TRIZOLReagent提取金仓鼠神经管总RNA;用LD PCR法反转录合成双链cDNA;经蛋白酶K消化、SfiⅠ酶切、 CHROMASPIN 400柱分离去除小于500bp的片段;将cDNA与载体λTriplEX2按一定比例连接;经体外包装,建立 cDNA的噬菌体表达文库。 结果 cDNA文库容量为1.5×106pfu ml,重组率为99%,平均插入片段大于1kb。 结论 我们初步构建的金仓鼠神经管cDNA文库为高效的cDNA文库。
Objective To construct a cDNA library of golden hamster neural tube to screen for the genes involved in the development of neural tube and the occurrence of neural tube defects. Methods Total RNA of golden hamster was extracted by TRIZOLReagent. Double-stranded cDNA was reverse transcribed by LD PCR. The digested cDNA was digested with proteinase K and digested with SfiI, and the fragment less than 500 bp was removed by CHROMASPIN 400 column. The cDNA was ligated with λTriplEX2 Ligation; in vitro packaging, cDNA phage expression library. Results The capacity of cDNA library was 1.5 × 106 pfu ml, the recombination rate was 99%, and the average inserted fragment was more than 1kb. Conclusion Our initial construction of the golden hamster neural tube cDNA library is a highly efficient cDNA library.