李ISSR反应体系的优化

Optimization on ISSR analysis system of plum

以李品种杏梅(hybrid)和斯顿莱(Prunus domestica)为试材,研究了PCR反应体系的主要成分及退火温度对李ISSR扩增结果的影响。结果表明:在20μl的反应体系中,模板DNA含量对扩增结果影响不大,在10-80 ng均能得到较好的扩增;dNTP用量对扩增无明显影响;而Primer、Mg2+的最适浓度分别为0.25/μmol/L、0.25 mmol/L;Taq酶在0.5-1.0 U均能得到好的扩增条带;退火温度在50-52.1℃范围内均能得到清晰的条带。

The factors which affected the ISSR(inter-simple sequence repeat) results of plum were studied. By adjusting tempkte DNA concentration, Mg2 + concentration, Primer concentration,dNTP and Taq Polymerase contents, and annealing temperature, the PCR amplification conditions were optimized.The results show that the optimized content containing 0.25μmol/L Primer,2.5mmol/L Mg2+,1U Taq Polymerase in 20μl system, and the optimized annealing temperature is between 50℃ and 52. 1℃.The optimal experiment conditions were as follow: 20μl system containing 20-80 ng, template, 0.25-0.3 μmol/L Primer,2.5 - 3.0 mmol/L Mg2+,0.2mmol/L dNTP, 1U Taq Polymerase,with a MJ thermal cycle;the optimal amplification program was 1 cycle of 5min at 94℃;40 cycles of 45 s at 94℃,45 s at 5℃-52. 1℃,and 1.5 min at 72℃; 1 cycle of 7 min at 72℃, using block control style.