摘要
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1．0U，引物浓度为10pmol／μl，基因组DNA浓度为50ng／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Mg^2＋）为2．0μl；dNTPs（各2．5mmol／L）为2．0μl；Taq酶浓度为1．0U，引物浓度为10pmol／μl，基因组DNA浓度为50ng／μl；超纯水为13．8μl。PCR扩增条件为：97℃预变性7min，94℃ 1 min，36℃退火1min，72℃ 2 min，45个循环后，在72℃延伸10min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2-3V／cm电压降稳压电泳3．0．3．5h。
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 hand could be obtained. The optimumized RAPD reaction system had a total volume of 20.0 μl,10 × Buffer （ Mg^2 ＋ included）2.0 μl,dNTPs（2.5 mmol/L respectively ）2.0 μl,Taq polyase concentration 1.0 U,pfimer concentration 10 pmol/ μl, genome DNA concentration 50 ng/μl, and extrame pure water 13.8 p.l. The amplification 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 ampLieon was tested through 1.4% agarose gel eleetrophoresis ,and after sample application,electrophoresis for 3.0 - 3.5 h under 2 - 3 v/era voltage.
出处
《山西农业科学》
2007年第6期103-105,共3页
Journal of Shanxi Agricultural Sciences
