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作者从一例人高转移型原发性胃癌组织及其对应的正常胃粘膜中分别提取总RNA和分离多聚(A)~+RNA,进而合成cDNA。cDNA第一条链合成效率前者为58.8%,后者为29.8%;第二条链合成效率前者为98.3%,后者为93.3%。cDNA分子大小在0.5～8Kb之间。cDNA接上含有NotI切点和EcoRI粘性末端的接头,重组到经EcoRI酶解5端脱磷酸化的pT7T318U质粒上,转化大肠杆菌NM522,构建了二个cDNA文库,文库的容量分别为1.2×10~5与1.0×10~5重组子,重组率均为80%,克隆效率分别为2.4×10~6克隆/lμg cDNA与2.0×10~6克降/lμg cDNA,插入片段的大小在0.5Kb～4Kb之间。
The authors extracted total RNA and isolated poly (A) ~ + RNA from a human highly metastatic primary gastric cancer tissue and its corresponding normal gastric mucosa, respectively, to synthesize cDNA. The first strand synthesis efficiency of the former was 58.8%, while the latter was 29.8%. The second strand synthesis efficiency was 98.3% and the latter 93.3%. The molecular size of cDNA is between 0.5 ~ 8Kb. The cDNA was ligated with a linker containing a NotI cleavage point and an EcoRI sticky end and recombined into the pT7T318U plasmid dephosphorylated by EcoRI digestion and transformed into E. coli NM522 to construct two cDNA libraries with capacities of 1.2 × 10 ~ 5 and 1.0 × 10 ~ 5 recombinants with the recombination rate of 80%. The efficiency of cloning was 2.4 × 10 ~ 6 clone / lμg cDNA and 2.0 × 10 ~ 6 g / lμg cDNA, respectively. The size of insert was 0.5Kb ~ 4Kb between.