水稻基因组DNA的RAPD扩增研究

Study on RAPD Reaction System for the Rice

通过对水稻RAPD反应条件比较研究,建立了对除草剂敏感致死水稻RAPD的最适反应体系和反应扩增程序,即在25μL反应体系中,含10mmol LTris＿HCl(pH8 0),10mmol LKCl,8mmol L(NH4)2SO4,0 05%NP＿40,2 0mmol LMgCl2,0 1mmol L(each)dTNPs,20ng引物,20ng基因组DNA,1 5U的Taq酶。反应扩增程序为:94℃变性2min;扩增40个循环,每循环包括:94℃变性10s,40℃退火30s,72℃延伸1 5min;最终延伸5min。

The optimal reaction system was constracted by studying the factors affecting the rice RAPD reaction.The system contained 10 mmol/L Tris-HCl (pH 8.0), 10 mmol/L KCl, 8 mmol/L (NH_4)_2SO_4, 0.05% NP-40, 2.0 mmol/L MgCl_2, 0.1 mmol/L dTNPs for each nucleotide, 20 ng pirmers, 20 ng rice DNA, and 1.5 U Taq polymerase in total 25 μL. The process of reaction was 95 ℃ for 120 s in the beginning, then 94 ℃ for 10 s, 40 ℃ for 30 s, 72 ℃ for 90 s, for 40 cycles, 72 ℃ for 300 s in the end.