鼠尾草属植物基因组DNA提取及RAPD反应条件优化

DNA Extraction from Salvia and Optimization of RAPD Reaction Condition

以鼠尾草属植物叶片为材料,提取基因组DNA,并对RAPD反应条件进行了系统优化。结果表明,采用核DNA法提取的DNA质量较高,适宜于RAPD分析;RAPD扩增最佳反应体系为:20μl反应体系中,10×buffer2.0μl,模板DNA20ng,Mg2+浓度2.0mmol/L,引物浓度0.6μmol/L,dNTPs浓度0.2mmol/L,Taq酶1.0U。扩增反应程序为:94℃预变性5min,94℃变性1min,36℃退火1min,72℃延伸2min;40个循环;72℃后延伸10min,4℃保存。

Used fresh leaves of Salvia as materials. The method of genomie DNA extraction and the optimization of RAPD reaction condition were studied in Salvia. The result showed that the high-quality genomie DNA was obtained by the nuclear DNA method and was sutiable for RAPD study. The optimal PCR system for RAPD analysis was as follows： total volume 20 μl, 2.0 μl 10xbuffer, 20 ng template DNA, 2.0 mmol/L Mg^2＋, 0.6 μmol/L Primer, 0.2 mmol/L dNTPs, TaqDNA 1.0 U. The program of amplifying reaction was as follows： After pre--denaturing at 94℃ for 5 min, under the condition of denaturing at 94℃ for 1 rain, annealing at 36℃ for 1 rain, extension at 72℃ for 2 min, amplifying for 40 cycles, extension at 72℃ for 10 min at last, stop at 4℃.