欧洲甜樱桃RAPD反应体系的建立与优化

以欧洲甜樱桃萨米托品种为研究对象，利用改进的CTAB法提取基因组DNA，并通过单因素多水平梯度试验，筛选了模板、Mg2＋、TaqE、dNTPs和随机引物的浓度及用量，建立了欧洲甜樱桃RAPD技术扩增体系．研究结果表明，在25出反应体系中，各组分最佳的浓度分别为：10×Buffer2．5μl，2．5mmol·L^-1Mg2＋2．5μl，Taq E0．06U·μl^-1，0．2mmol·L^-1dNTPs，0．2μmol·L^-1Primer，模板DNAl．2ng·μl^-1．PCR循环程序为：94℃预变性4min。94℃变性40s，36℃退火45s，72℃延伸1min，共36个循环，最后72℃延伸7min．

Establishment and Optimization on RAPD Analysis System of European Sweet Cherry

An improved DNA extraction protocol with CTAB was developed for European sweet cherry. The genomic DNA was employed to establish optimization of RAPD. The RAPD system for European sweet cherry （Prunus avium L. ） was optimized including program template, primer, dNTPs,Taq polymerase and Mg2＋ . Through ladder experiments, the reliable RAPD analysis system is established. The total reaction volume is 25μl and reaction mixture consists of 5/A 10 ×Buffer, 1.2ng·μl^-1 Template DNA, 0.2mmol· L^-1 dNTPs, 2.5mmol· L^-1 Mg2＋ , 0. 2μmol· L^-1 random Primer and 0.06 U·μl ^-1 Taq E. RAPD program is 4 min at 94℃ for predenaturation, then 36 cycles of 40s at 94℃ for denaturation, of 45s at 36 ℃ for annealing,of lmin at 72℃ for extension,finally extension at 72℃ for 7min.