摘要
利用Trizol一步法从柔嫩艾美耳球虫孢子化10h卵囊提取总RNA。利用poly(A)QuikmRNAIsolationkit分离纯化了mRNA。采用cDNASynthesisKit合成cDNA,以XR-λZAP为载体,构建了cDNA表达文库,经蓝白斑筛选,其重组率为98%,滴度为0·9×106pfu/mL,扩增后滴度为1×109pfu/mL。随机取10个克隆,提取λDNA后,经PCR鉴定,插入片段长度大于0·4Kb。该cDNA表达文库的构建为下一步功能基因的筛选等奠定了良好基础。
Total RNA of Eimeria tenella was extracted by the Trizol procedure from oocysts sporulated for 10h and analyzed by 1% electrophoresis. The mRNA was isolated with the poly （A） Quik mRNA Isolation kit. The cDNA were synthesized by a cDNA Synthesis Kit, then cloned into the XR-λZAP vector. The recombination fraction was 98% by blue-white spot screening. The titre was 0.9 × 10^6 pfu/mL, that after amplification was 1 × 10^9 pfu/mL. The size of insert cDNA was larger than 0.4Kb by PCR with λDNA of 10 random clones.
出处
《寄生虫与医学昆虫学报》
CAS
2005年第3期143-145,共3页
Acta Parasitologica et Medica Entomologica Sinica
基金
国家自然科学基金农业倾斜项目(No.30170696)
