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以高粱细胞质雄性不育系和保持系 A1Tx6 2 3 A/B,A2 V4 A/B为材料 ,考察了 Mg2 + ,Taq酶 ,d NTP,引物 ,模板等的用量、循环参数及不同的 PCR仪对随机扩增多态性 DNA(RAPD)反应的影响。结果发现 :在 2 5 μL体系中 ,Mg Cl2 2 .0 mmol/L,Taq酶 0 .75 U(1 U=1 μmol/min) ,d NTP0 .1 5mmol/L,引物 1 6 .5 ng,模板 3 0 ng,明胶 0 .0 0 1 % ,KCl5 0 mmol/L,Tris1 0 mmol/L(p H8.3 )为最佳反应混合物组合。对 Peltier Thermal Cycler 2 0 0而言 ,94℃预变性 5 min,94℃变性 1 min,3 5℃退火 1 min,72℃延伸 2 min,最后 72℃保温 5 min,共计 40个循环为最佳扩增条件 ;对 Perkin ElmerCetus DNA Thermal Cycler- 480而言 ,采用 94℃预变性 3 min后 ,前 3个循环 ,94℃变性 1 min,3 5℃退火 1 min,72℃延伸 2 min,后 3 7个循环 ,94℃变性 3 0 s,3 6℃退火 45 s,72℃延伸 1 min,最后72℃保温 5 min的循环程序可获得理想扩增。并阐述了优化 RAPD实验体系的设计原则。
The sorghum cytoplasmic male sterile lines and maintainer lines A1Tx6 2 3 A / B and A2 V4 A / B were used as materials to investigate the effects of Mg2 +, Taq enzyme, dNTP, primers, templates, On the effects of random amplified polymorphic DNA (RAPD) reactions. The results showed that the optimal conditions were as follows: MgCl 2 2.0 mmol / L, Taq enzyme 0 .75 U (1 U = 1 μmol / min), dNTP 0.15 mmol / L, 30 ng gelatin, 0 0 01% gelatin, 0 mmol / L KCl5, and 0 mmol / L Tris1 (p H8.3) were the best combination of reaction mixtures. For Peltier Thermal Cycler 200, the initial denaturation at 94 ° C for 5 min, denaturation at 94 ° C for 1 min, annealing at 35 ° C for 1 min, extension at 72 ° C for 2 min and final incubation at 72 ° C for 5 min resulted in a total of 40 cycles For the Perkin ElmerCetus DNA Thermal Cycler-480, the first three cycles of denaturation at 94 ° C for 3 min followed by denaturation at 94 ° C for 1 min, annealing at 35 ° C for 1 min, extension at 72 ° C for 2 min and then 3 Seven cycles of denaturation at 94 ° C for 30 s, annealing at 36 ° C for 45 s, extension at 72 ° C for 1 min, and final incubation at 72 ° C for 5 min gave ideal amplification. And elaborates the design principle of optimizing RAPD experiment system.