佛手RAPD扩增系统的建立

Establishment of RAPD amplification system in Bergamot

试验了模板DNA、引物、镁离子和Taq酶的浓度对佛手RAPD分析结果的影响,从而建立了适合佛手RAPD扩增的系统.具体条件为:25μL反应体系中模板DNA100ng(4ng μL),MgCl23.0～5.0mmol L,dNTP200μmol L,10 merPrimer0.2μmol L及1UTaqPolymerase.扩增程序为:93℃120s;之后,93℃60s,35℃60s,72℃120s,进行42个扩增循环;最后72℃延伸600s.

In this paper,the influencing factors on RAPD experiment were analyzed,such as the template concentration,the Mg^(2+)concentration,the primer concentration,the Taq polymerase concentration,and so on.The results indicated that each 25 μL amplification reaction was consisted of 100 ng template DNA,3～5 mmol/L Mg^(2+),200 μmol/L dNTP,0.2 μmol/L 10-mer primer,1 U Taq polymerase.The amplification procedure conditions were predenature at 93 ℃ 180 s followed by denature at (93 ℃) 60 s,annealing 35 ℃ for 60 s,extension 72 ℃ for 120 s,cycling for 42 times,last extension (300 s)and then the optimum procedure for Bergamot DNA amplification was obtained.