摘要
以裸果木新鲜叶片为材料,采用改进CTAB法提取基因组DNA,在参考一般RAPD分析反应程序的基础上,研究了裸果木RAPD分析过程中的影响因素Taq酶、Mg2+、dNTP、引物、模板DNA浓度、变性时间等,建立了适于裸果木RAPD反应的PCR体系:模板DNA为4ng/μL,引物浓度为0.3μmol/L,dNTP浓度为0.5mmol/L,Mg2+浓度为2.0mmol/L,Taq酶用量为1U,ddH2O补足25μL;94℃预变性5min,94℃变性45s,36℃退火40s,72℃延伸1min,共30个循环,最后72℃延伸10min,扩增结果稳定。
To study the RAPD optimal reaction system of G.przewalskii and provide the research basis for RAPD amplification.The DNA templates extracted from fresh leaves by CTAB methods,such factors impacted on RAPD analysis as Taq enzymes,Mg2+,dNTP,primer,concentration of DNA template,time of degeneration and temperature were studied,and PCR systems fitted for RAPD reaction of G.przewalskii were established.The reactions were performed in a volume of 25 μL containing 2.5μL 10×Buffer,2.5 mmol/L Mg2+,Taq DNA ploymerase 1U,0.5 mmol/L dNTPs,0.3μmol/L Primer,and 4.0 ng/μl template DNA.PCR program was 5 min at 94℃ for predenaturation,then 30 cycles of 45 s at 94 ℃ for denaturation,40 s at 36 ℃ for annealing,1 min at 72 ℃ for extension,finally extension at 72 ℃ for 10 min.
出处
《草原与草坪》
CAS
2012年第1期7-11,共5页
Grassland and Turf
基金
公益性农业行业专项-牧区生态高效草原牧养技术模式研究与示范(201003061)资助
关键词
裸果木
RAPD
反应体系
优化
Gymnocarpos przewalskii
RAPD
reaction system
optimization
