獭兔RAPD反应体系优化研究

对獭兔RAPD体系中Taq DNA聚合酶浓度、引物浓度、基因组DNA浓度进行研究。结果表明:Taq酶浓度为1.0 U,引物浓度为10 pmol/μl,基因组DNA浓度为50 ng/μl时,可获得清晰、稳定性好的条带。优化的RAPD反应体系为:总体积为20.0μl。10×Buffer(含Mg2+)为2.0μl;dNTPs(各2.5 mmol/L)为2.0μl;Taq酶浓度为1.0 U,引物浓度为10 pmol/μl,基因组DNA浓度为50 ng/μl;超纯水为13.8μl。PCR扩增条件为:97℃预变性7 m in,94℃1 m in,36℃退火1 m in,72℃2 m in,45个循环后,在72℃延伸10 m in结束,4℃保存。PCR扩增产物通过1.4%的琼脂糖凝胶电泳检测。点样后,以2~3 V/cm电压降稳压电泳3.0~3.5 h。

Optimumization of RAPD Reaction System in Rex Rabbit

In present paper,concentrations of Taq DNA polyase,primers and genome DNA were studied.The results showed that,when concentrations of Taq polyase was 1.0 U,of primer 10 pmol/μl and of genome DNA 50 ng/μl,clear and good stable band could be obtained.The optimumized RAPD reaction system had a total volume of 20.0 μl,10×Buffer(Mg2+ included)2.0 μl,dNTPs(2.5 mmol/L respectively)2.0 μl,Taq polyase concentration 1.0 U,primer concentration 10 pmol/μl,genome DNA concentration 50 ng/μl,and extrame pure water 13.8 μl.The amplitication conditions were predonaturation for 7 minutes under 97 ℃ and for 1 minuter under 94 ℃,annealing for 1 minute under 36 ℃ and for 2 minutes under 72 ℃,after 45 circulations doing extension for 10 minutes under 72 ℃ and then ceased,and preserved under 4 ℃ PCR amplicon was tested through 1.4% agarose gel electrophoresis,and after sample application,electrophoresis for 3.0~3.5 h under 2~3 v/cm voltage.