摘要
为了研究舰船有害气体、噪声、磁场等特殊环境下人脑特异性基因表达的变化情况 ,构建了一个 2 0周人胎脑cDNA文库 ,实验从胎脑组织中抽提总mRNA后 ,经过一系列酶反应后合成cDNA ,分级分离柱除去小片段后克隆到λgt1 0载体 ,转染宿主菌E .coli.C6 0 0hf1后文库的包装效率为 4 .6× 1 0 6pfu/μg ,cDNA平均插入片段大于 1 .
To construct a cDNA expression library from 20-week human embryonic brain, 16.6mg total RNA and 192ug poly(A)+ mRNA have been successively obtained from the 20-week human embryonic brain using 'single-step method' and by chromatography on oligo-(dT) cellulose. With reverse transcriptase (M-MLV), 10μg mRNA was synthesized into 4.4μg blunt cDNA. After the cDNA was ligated to EcoR I adapter and purified by fractionation, 400ng cDNA of about 100bp-8.0kb was collected, of which 50ng cDNA was inserted into λgt11 phage particles. A cDNA library containing 4.6×10 6 recombinants has been constructed. The capacity of this library and the size of cDNA is suitable for futher study.
出处
《氨基酸和生物资源》
CAS
2001年第4期17-20,共4页
Amino Acids & Biotic Resources
