七彩鲑RAPD反应体系的构建及优化

Optimization for RAPD reaction system in Salvelinus fontinalis

以七彩鲑尾鳍为试验材料,对影响七彩鲑随机扩增多态性DNA(RAPD)扩增体系的重要参数进行了优化,建立了七彩鲑RAPD的最适反应体系.七彩鲑RAPD的最佳反应体系为:反应体系25μL,模板DNA的质量浓度4ng/μL,Mg2+浓度2.0mmol/L,dNTP浓度0.5mmol/L,引物浓度0.5μmol/L,Taq酶用量0.04U/μL.扩增程序为94℃预变性4min,94℃变性45s,36℃退火40s,72℃延伸1min,共30个循环,最后72℃延伸8min.

The caudal fin of Salvelinus fontinalis was used in this study to optimize the several factors applied in RAPD reaction system.The RAPD reaction system was established.The results showed that the optimum reaction system was performed in a volume of 25 μL containing 2.0 mmol/L Mg2＋,0.5 mmol/L1 dNTPs,0.5 μmol/L1 primer,4 ng/μL1 template DNA,and 0.04 U/L1 Taq DNA polymerase.The PCR program was 4 minutes at 94 ℃,for predenaturation,30 cycles of 45 second at 94 ℃ for de-natureation,40 second at 36 ℃ for annealing,1 minute at 72 ℃ for extension,finally extension at 72 ℃ for 8 minutes.