金弹总DNA提取及RAPD体系优化

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。

Total DNA Extraction and RAPD System Optimization of Fortunella crassifolia

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℃.