云南松胚乳DNA提取与ISSR-PCR反应体系的建立

以云南松胚乳为试验材料,用改进的SDS法进行DNA的提取,并对其纯度、浓度及产率进行检测,结果表明：DNA扩增效果良好,完全能满足进一步试验的需要 采用单因子试验分析法分别研究模板DNA浓度、引物浓度、Mg2＋浓度、TaqDNA聚合酶用量、dNTP浓度、退火温度对反应体系的影响,建立并优化云南松ISSR-PCR 20μL反应体系：buffer（10 mmol/L Tris-HCl,50 mmol/L KCl,0.1%Triton X-100 pH 9.0）,0.3μmol/L引物,1.5 m mol/L MgCl2,0.5 U TaqDNA聚合酶,0.2 mmol/L dNTPs,30 ng模板DNA,引物（GA）8的最适退火温度为52.5℃。

Endosperm DNA Extraction from Pinus yunnanensis Seeds and Establishment of the ISSR-PCR Reaction System

Improved SDS method was applied to extract DNA from the endosperm of Pinus yunnanensis seeds, and the purity, concentration and output of the DNA were tested. The results showed that the DNA could be well amplified, which was fully able to meet the demand of further experiments. At the same time, a single factor exper- imental analysis was applied in this experiment to study the DNA template concentration, concentrations of the primer, Mg2＋ , dNTP, Taq DNA polymerase dosage, and the influence of annealing temperature on the reaction system. The ISSR-PCR reaction system for P. yunnanensis tree species was established and optimized as the total volume of the PCR reaction system was 20μL, with 10 times of buffer （ 10 m mol/L Tris-HC1, 50 m mol/L KCI, 2 μL of 0. 1%TritonX-100 pH 9.0）, 0. 3μ mol/L primer, 1.5 m moL/L MgCl2, 0. 5 U TaqDNA polymerase, 0. 2 m mol/L dNTPs, 30 ng of template DNA. The optimal annealing temperature for primer （GA） s was 52. 5 ℃.