苹果基因组RAPD反应体系影响因子的优化

Optimization of Factors Affecting RAPD Amplification in Apple Genome

分析了影响苹果基因组RAPD扩增的几种因素,包括模板DNA、Mg2+、dNTPs和引物的浓度、以及Taq酶的用量等,并提出了一个最佳方案,即25μl总反应体系中含DNA模板40～60ng、Mg2+2.0mmol/L、dNTPs0.2mmol/L、引物0.45μmol/L、Taq酶1.2U。本研究的PCR反应程序为:94℃预变性4min,然后按94℃变性30s,37℃退火35s,72℃延伸90s进行40个循环,最后72℃延伸5min。

In this study, several factors, including concentrations of DNA, Mg2+, dNTPs, primer and TaqE, affecting RAPD amplification in apple genome were analyzed and a better system was promoted: the total reaction volume was 25μl which contained DNA template 40～60ng, TaqE 1.2U, MgCl2 2.0mmol/L, dNTPs 0.2 mmol/L and ten base random primer 0.45μmol/L. The PCR amplification procedure used in this study was as follows: pre-denature at 94℃ for 4 mins, and then 94℃ 30s,37℃ 35s,72℃ 90s,for 40cycles, finally extended at 72℃ for 5mins.