摘要
利用Trizol法从津田芜菁中提取总RNA,分离纯化富含Poly(A)+的mRNA,反转录合成双链cDNA,并以λ-ZAP为载体,构建了津田芜菁cDNA文库.以XL1-Blue为受体菌,测定原始文库的滴度为4.3×106pfu/mL,重组率为96%,扩增后文库总滴度为6×1010pfu/mL.对随机提取的质粒进行PCR鉴定,表明插入片段大多分布在0.5～2.0kb之间.文库质量鉴定结果表明,构建的津田芜菁直根皮cDNA文库具有较好的库容量、较高的重组率以及较大的插入片段.对3020个克隆的序列测定表明,获得可用序列2718个,测序成功率为90%,首批共获得652个芜菁直根皮ESTs.
Total RNA was isolated from the peel of“Tsuda”taproot by using Trizol method, mRNA rich in Poly(A) + was separated and purified, and then the double-strand c DNA was synthesized. After size fractionation, the ds-cDNA was ligated to λ-ZAP vector and recombinant bacteriophages were packaged. The cDNA library was const ructed and amplified with XL1-Blue as receptor bacterium. The titer of the origi nal library was 4.3×10 6pfu/mL with a recombinant rate of 96%, and the amplifi ed titer was 6×10 10 pfu/mL. PCR amplification of the random plasmid sugges ted that the inserted cDNA fragments ranged from 0.5kb to 2.0kb. The apprais al of quality indicated that the cDNA library had higher titer and recombinant r ate as well as large inserted fragments. Totally, 3020 clones were sequenced, an d 2718 useful sequences were obtained with the successful ratio of 90%. The firs t batch of 652 valid ESTs was generated.
出处
《东北林业大学学报》
CAS
CSCD
北大核心
2005年第2期18-19,37,共3页
Journal of Northeast Forestry University
基金
国家自然科学基金项目(30170785)
