苜蓿基因组DNA提取和RAPD反应条件优选

采用CTAB法、N2-SDS法、SDS法和改进的CTAB法,提取苜蓿基因组DNA,比较其分离效果.结果表明,改进的CTAB法为最佳提取法.对RAPD反应程序中的一些重要参数进行摸索和优化试验,建立适合苜蓿RAPD分析应用的体系组成为:反应液总体积25μL,14ng/μL模板DNA,2.5μL10x反应缓冲液,10碱基引物浓度0.1μmol/L,TaqDNA聚合酶1U,dNTP浓度0.3mmol/L,Mg²⁺浓度2.5mmol/L.PCR反应循环为:94℃4min,36℃30s,2℃1min;94℃30s,36℃30s,2℃1min,45个循环;2℃延伸10min.

Optimization of Alfalfa Genomic DNA Extraction And RAPD Conditions

Four different methods were tested in isolation of alfalfa (Medicago sativa) genomic DNAs.The results show that the reformed CTAB is more effective.Based on the genomic DNA extracted from alfalfa,essential factors affecting the result of RAPD assay were tested and compared.An optimal reaction system suitable for the assay and usage of RAPD in alfalfa was established.This optimal of 25 μL reaction solution contained 2.5 μL 10x buffer,2.5 mmol/L MgCl 2,0.3 mmol/L each dNTP,0.1 μmol/L random primer,174 ng/μL template DNA,1 U Taq polymerase.The reaction program was devised for one cycle at 94℃ 4 minutes,36℃ 30 seconds,72℃ 1 minute,which is followed by 45 cycles,each with 30 seconds at 94℃,30 seconds at 36℃,1 minutes at 72℃,and a final extension at 72℃ for 10 minutes.