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以改进的CTAB高盐法提取油茶嫩叶总DNA,进行简单重复序列间扩增(ISSR)分析,分别测试了退火温度、模板DNA浓度、M g2+浓度、dNTP s浓度、T aqDNA聚合酶用量和是否加入去离子甲酰胺对反应结果的影响.油茶ISSR分析的最适反应体系为:在20μL PCR反应体积中,含20 ng模板DNA,2.5 mm o l.L-1M gC l2,0.2 mm o l.L-1dNTP s,1UT aqDNA聚合酶,0.5pm o l.μL-1引物,1%去离子甲基酰胺.扩增程序为:94℃预变性2 m in;94℃变性30 s,52℃退火30 s,72℃延伸90 s,反应38个循环;最后在72℃延伸7 m in.
The total DNA of young leaves of Camellia oleifera was extracted with improved high-salt method of CTAB, and the ISSR analysis was carried out. The annealing temperature, template DNA concentration, M g2 + concentration, dNTPs concentration, T aq DNA polymerase dosage and The optimal reaction system for ISSR analysis of Camellia oleifera was as follows: 20 ng of template DNA, 2.5 mmole of IL-1M gC12, 0.2 mmo lL-1dNTP s, 1UT aqDNA polymerase, 0.5 pmol·L-1 primer, 1% deionized methylamide.The amplification program was: pre-denaturation at 94 ° C for 2 min, denaturation at 94 ° C for 30 s, annealing at 52 ° C for 30 s, Extension at 72 ° C for 90 s, reaction for 38 cycles; and finally extension at 72 ° C for 7 min.