金弹总DNA提取及RAPD体系优化

Total DNA Extraction and RAPD System Optimization of Fortunella crassifolia

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。

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 Mg^2＋, 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 Mg^2＋, 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 cvcle for 10 min at 72 ℃. storing at 4 ℃.