中国地鼠RAPD分析体系的优化

Study on RAPD Analysis System in Chinese Hamster

目的 优化中国地鼠RAPD分析体系。方法 以30条引物对山医群体中国地鼠的两个家系基因组DNA在各种不同条件下进行RAPD ,分析比较扩增结果。结果 在下列反应体系及温度循环参数下结果较好:在2 5 μl的反应体系中含有10×buffer 2 5 μl,dNTP 0 2mmol L ,Mg2 + 2 0mmol L ,引物0 2 μmol L ,Taq酶1 5U ,模板DNA 15ng ,扩增程序为94℃预变性3min后进入循环体系,94℃变性30s ,36℃退火5 0s ,72℃延伸1min ,4 0个循环后接7min延伸。结论 以上述反应体系及温度循环参数进行中国地鼠RAPD 。

Objective To optimize the RAPD procedure in Chinese Hamster. Method Thirty primers were used to amplify DNA in 2 genealogies of Chinese Hamster and then the results of the amplification were analyzed. Results Optimal reaction system was as follows: 25 μl of each PCR mixture containing 2.5 μl of 10×Taq reaction buffer, 0.2 mmol/L of each dNTPs, 2.0 mmo/L of Mg 2+, 0.2 μmol/L of primer, 1.5 U of Taq DNA polymerase and 15 ng of total DNA. The reaction condition was predenaturation at 94℃ for 3 min, then 40 cycles of denaturation at 94℃ for 30 s, annealing at 36℃ for 50 s and extension at 72℃ for 1 min, finally extension at 72℃ for 7 min. Conclusion The use of the above-mentioned reaction system and amplifying procedure for RAPD in Chinese Hamster can get stable results.