羊角槭基因组DNA提取及SRAP-PCR体系优化

Extraction of Genomic DNA from Acer yangjuechi and System Optimization of SRAP-PCR

采用CTAB法、SDS法、偏重亚硫酸钠法提取槭属中羊角槭基因组DNA,通过DNA含量测定、琼脂糖凝胶电泳检测对所提DNA质量进行分析。结果表明,3种提取方法中CTAB法最适合提取羊角槭基因组DNA,所得DNA的质量和纯度较高。以CTAB法提取的DNA为模板进行SRAP扩增反应,对SRAP反应体系的dNTPs浓度、Mg2+浓度、引物浓度3个主要影响因子进行筛选。获得羊角槭SRAP最优反应体系为:50μl的PCR体系中含有DNA模板100 ng、10×PCR Buffer(不含Mg2+)、Mg2+2.5 mmol/L、dNTPs 0.2 mmol/L、引物0.5μmol/L、TaqDNA聚合酶1.0 U。

To find a rapid and effective DNA extraction method for Acer, CTAB method, SDS method and Na2S2O5 method were compared. The DNA was detected by Eppdendorf Biophotometer and agarosegel electrophoresis. The results showed the CTAB method was much better than the others in extracting DNA of Acer yangjuechi, and which was suitable for SRAP-PCR analysis. The concentrations of dNTPs, Mg^2 ＋ and primers which affect the SRAP-PCR reactions were optimized in order to establish the SRAP molecular marker system in Acer. The optimum system was as follows： template DNA 100 ng, 10 x PCR Buffer （ Mg^2＋ free）, Mg^2＋2. 5 mmol/L, dNTPs 0. 2 mmol/L, primers 0. 5μmol/L, Taq DNA polymerase 1.0 U. The total volume of reaction was 50μl.