观赏桃ISSR-PCR反应体系的优化

Optimization for ISSR-PCR Reaction Conditions of Ornamental Peach

采用改良CTAB法从观赏桃满天红叶片中提取基因组DNA,通过单因素实验探讨了模板DNA、Mg2+、dNTPs和TaqDNA酶等条件对观赏桃ISSR-PCR扩增结果的影响,建立了ISSR-PCR扩增的最佳体系:25μl反应体系中包含10×Buffer2.5μl,模板DNA40ng,Mg2+浓度2.5mmol/L,引物浓度0.4μmol/L,dNTPs浓度0.4mmol/L,TaqDNA酶0.5U。利用所建立的体系对红叶桃、菊花桃和春艳等13份材料进行检验,其结果表明优化后的体系适合观赏桃的ISSR-PCR反应。

The improved method CTAB was used to extract DNA from leaves of ornamental peach mantianhong. The research discussed different components＇ effect to the DNA amplification, including program template, primer, dNTPs, Taq polymerase and Mg2＋ , through ladder experiments. The reaction conditions were optimized and then the optimal PCR system for ISSR analysis was established as follows,total volume 25μl,2.5μl 10 Buffer,40 ng template D NA,2.5 mmol/L Mg2＋,0. 4 mol/L Primer,0.4 mmol/L dNTPs,and 0. 5 U Taq DNA polymerase. The optimal PCR system was tested by 13 samples,showing that it performed satisfactorily on ornamental peach.