海南普通野生稻ISSR-PCR反应体系的优化

STUDY ON OPTIMIZATION FOR ISSR-PCR REACTION SYSTEM FOR WILD RICE IN HAINAN PROVINCE

通过不同浓度的Mg2+、模板DNA、dNTPs、Taq酶、引物、退火温度和循环次数的单因素试验,筛选海南普通野生稻最佳ISSR-PCR反应体系为:15μl反应体系中含2.0 mmol/L MgCl2,50 ng模板DNA,200μmol/L dNTPs,1.20 U Taq酶,1.5μmol/L ISSR引物,50 mmol/L KCl 10 mmol/L Tris-HCl(pH8.3).反应程序:94℃预变性5 min;94℃,1 min,52℃,1 min,72℃,2 min,35个循环;72℃延伸10min,4℃保存.

To optimize ISSR-PCR reaction system for wild rice in Hainan Province,the effects of seven factors Mg2+,dNTPs,Taq DNA polymerse,primer,T-DNA,annealing temperature and cycle were studied at six levels respectively.The results showed that optimum ISSR-PCR reaction system of wild rice was every 15 μL reaction system containing 2.0 mmol/L MgCl2,50ng template DNA,200μmol/L dNTPs,1.20U Taq DNA polymerse,1.5 μmol/L ISSR primer,50 mmol/L KCl,10 mmol/L Tris-HCl(pH 8.3).The above reaction mixture was denatured at 94℃...