柚ISSR-PCR反应体系的建立与优化

Establishment and Optimization of ISSR-PCR Reaction System in Citrus grandis(L.)

以柚类种质资源为材料,对影响其ISSR-PCR反应结果的模板浓度、Taq酶浓度、dNTPs浓度、引物浓度、循环次数进行了探讨.结果表明,25μL的反应体系为:20-80ng的DNA,0.2mM dNTPs,2.0mM MgCl2,1μM引物,1.5U Taq酶.PCR扩增程序为:94℃预变性4min,94℃变性40sec,48-58℃(视引物而定)1min,72℃延伸2min,35个循环,72℃延伸7min.

Factors affecting the ISSR-PCR analysis of Citrus grandis （L.） in this study, such as concentration of template DNA, TaqDNA polymerase, dNTPs, ISSR primer and the number of cycle, were studied. The results show that： in a total volume of 25 μL, the reaction system should contain 20 - 80ng DNA, 0.2raM dNTPs, 2. 0raM MgCl2, 0. 2μM primer and 1U Taq DNA polymerase. The amplification was after 1 cycle initial denaturation at 94℃ for 4 rain, followed by 35 cycles of 40s at 94℃, 1 rain at 48 - 58℃ （ differing on different primers）, 2 rain at 72℃, and final 7 rain extension at 72℃.