小干松SRAP-PCR反应体系的建立

Establishment of the SRAP-PCR Reaction System of Pinus contorta

以小干松针叶基因组DNA为模板,采用L16(45)正交实验设计,对SRAP-PCR反应体系中的Taq酶、Mg2+、dNTPs、模板DNA和引物5个因素在4个水平上进行优化。结果表明:小干松SRAP-PCR 20μL反应体系最佳组合为:Taq酶0.5U,Mg2+浓度2.5mmol/L,dNTPs浓度0.15mmol/L,模板DNA含量60ng,引物0.2μmol/L。使用12对SRAP引物,采用优化后的体系进行SRAP-PCR反应,表明优化的体系很好地满足了小干松基因组DNA进行SRAP的扩增要求。

To establish the optimal SRAP reaction system of Pinus contorta ,its genomic DNA was extracted as template and orthogonal experimental design L16 （4s） were used. Four levels of five factors （Taq polymerase,Mg2＋ ,dNTPs,DNA template and primer） were optimised. The results showed that according to the amplification results, an optimal and stable SRAP-PCR system for Pinus contorta was 0. 5 U Taq polymerase,2.5 mmol/I. Mg2＋ ,0. 15 mmol/L dNTPs,60 ng DNA template and 0. 2 μmol/L primer in total 20 μL reaction solution. 12 pairs SRAP primer were used in this optimized SRAP-PCR system, the results showed that this system could meet the requirement for SRAP amplification of Pinus contorta .