蜡梅花瓣基因组DNA提取及RAPD-PCR反应体系优化

摘要

The high purity and quality genomic DNA of the petals of Chimonanthus praecox cultivars were extracted by the modified CTAB method, and different gradients were set for each PCR reaction factor to do experiment.The optimal RAPD-PCR system was established with orthogonal design.The results showed that a total volume of 20 μ L RAPD-PCR system were consisted of 1 × buffer, 1.0 U Taq DNA polymerase, 2.0 mmol/L MgCl2, 0.15 mmol/L dNTPs, 0.5 μ mol/L primer and 30 ～ 40 ng tempalte DNA.The suitable procedure was one cycle denaturing at 94 ℃ for 3 min, 38 cycles involving denaturing at 94 ℃ for 30 s, annealing at 38 ℃ for 30 s, extending at 72 ℃ for 90 s and one cycle extending at 72 ℃ for 7 min,and later kept at 4 ℃.%以蜡梅花瓣为试材,用改良CTAB法提取基因组DNA,并设置不同浓度梯度对每个PCR反应因子进行相应试验,结果表明,改良CTAB法提取蜡梅花瓣基因组纯度高、质量好.并采用正交试验设计的方法,对蜡梅RAPD-PCR条件进行了优化,结果表明,最佳的蜡梅RAPD-PCR反应体系(20μL)为:1×buffer,1.0 U TaqDNA聚合酶,2.0 mmol/LMgCl2,0.15 mmol/LdNTPs,0.5μmol/L引物和模板DNA30～40ng;适宜的扩增条件为:94℃预变性3 min;94℃变性30 s,38℃退火30 s,72℃延伸90s,38个循环;72℃延伸7 min,4℃保存.