美花石斛RAPD-PCR反应体系的建立

Study on the RAPD-PCR Amplification System of Dendrobium loddigseii

目的建立美花石斛基因组DNA的RAPD-PCR最佳反应体系。方法在提取纯化美花石斛基因组DNA的基础上,利用PCR扩增技术和方法,对Mg2+,dNTP,模板DNA,引物,Taq聚合酶等反应条件进行优化。结果反应体系的最佳条件是总体积为25μl,其中Mg2+浓度1.2 mmol.L-1,Taq聚合酶1.2U,引物浓度0.3 mmol.L-1,模板DNA 46 ng和dNTPs 160 mmol.L-1;PCR扩增程序为94℃预变性3 min,94℃变性50 s,40℃退火1 min,72℃延伸2 min,循环40次,72℃延伸5 min。结论该反应体系具有经济,简便以及扩增条带较清晰而稳定等特点。

Objective To establish the best reaction condition of RAPD - PCR amplification system of Dendrobium loddigesii. Methods Some reaction condition was optimized by amplifying technology and method of PCR based on isolating genomic DNA from D. loddigesii. Results The best amplification system for D. loddigesii was as follows ： total reaction volume 25 μl including 1.2 mmol · L^-1 Mg^2＋ ,1, 2 unite Taq polymerase, 0. 3 mmol · L^-1 random primer, 46 ng template DNA and 160 mmol · L^-l dNTPs; the amplification procedure conditions were pre -denature at 94℃ for 3 min followed by denature at 94℃ for 50 s, annealing at 40℃ for lmin, extension at 72℃ for 2 min,cycling 40 times, last extension at 72℃ for 5 min. The PCR amplification bands for five individuals of this species were very stable and clear in this RAPD - PCR amplification system. Conclusion This amplification system is very economical, convenient, and the band is clear and stable.