Abstract: Three different methods were used for total DNA extraction from fresh leaves of Fortunella crassifolia,respectively.The results showed that the most effective method for RAPD analysis was improved CTAB Ⅱ protocol.Four influencing factors on RAPD-PCR,including Mg²⁺,dNTP,primer and DNA template concentration were tested using single factor design and uniform design.A suitable RAPD-PCR system for F. crassifolia was established as follows:20 μl PCR solution,2.5 mmol/L Mg²⁺,0.25 mmol/L dNTP,0.20 μmol/L primer,1.0 ng/μl DNA template,1 U Taq DNA polymerase and 10×buffer.The RAPD-PCR was programmed by a cycle for 3 min at 94℃,followed by 45 cycles for 45 s at 94℃,60 s at 37℃ and 120 s at 72℃,and finally by a cycle for 10 min at 72℃,storing at 4℃.

Key words: Fortunella crassifolia, DNA extraction, RAPD

摘要： 以金弹叶片为材料,研究其总DNA提取方法及RAPD-PCR条件。结果表明,用改良CTAB法Ⅱ提取的DNA适于RAPD分析;优化的金弹RAPD-PCR体系为:反应体积20μl,Mg²⁺2.5 mmol/L、dNTP 0.25 mmol/L、引物0.20μmol/L、模板DNA 1.0 ng/μl和1 U Taq DNA聚合酶。相应的扩增程序为:94℃预变性3 min;94℃变性45 s,37℃复性60 s,72℃延伸120 s,循环45次;72℃延伸10 min,4℃结束。

关键词: 金弹, DNA提取, RAPD