剑麻DNA高效提取及RAPD反应体系优化

Extraction of Genomic DNA and Optimization of RAPD Reaction System in Sisal

针对剑麻组织中多糖、多酚和次生物质含量高的特点,对其基因组DNA提取方法进行研究。比较了SDS法、CTAB法提取基因组DNA的效果,结果分析表明:CTAB法提取效果较佳,A260/A280为1.8左右,A260/A230大于2.0,电泳检测基因组DNA纯度和完整性较好。后基于基因组DNA水平,对RAPD反应体系中镁离子浓度、dNTP浓度和退火温度三个重要的影响因子进行优化,得出在25μl反应液中,Mg2+浓度为2.0mmol·L-1,酶为1.0U,引物为0.1"mol·L-1,dNTP为0.2mmol·L-1,37℃退火温度扩增40个循环效果较佳。

Study on extraction of high quality DNA from sisal was carried out in allusion to high levels of polysaccharides and polyphenols combined with nucleic acid. We compared the methods of SDS and CTAB extracting DNA from sisal, the results showed that CTAB extraction method is more effective, the ratio of A260/A280 of extracted sisal DNA is 1.8, the ratio of A260/A230 is more than 2.0. The extracted DNA is very pure and integrity. Meanwhile, reaction system of RAPD was optimized basing on the level of DNA. The reaction program of amplification was Mg^2＋ 2.0 mmol·L^-1, Taq 1.0U, dNTP 0.2mmol·L^-1, random primer 1.0 μmol·L^-1 in 25μl reaction system.