不同种源韦塔桉RAPD反应条件优化研究

Study of RAPD reaction conditions on Eucalyptus wetarensis

对13个韦塔桉种源的RAPD反应条件,即PCR反应体系、PCR扩增条件等因素进行了研究。结果表明,最佳PCR体系为:DNA模板为10.0μL、双蒸水4.0μL、10×缓冲液2.0μL、25mMMgCl21.5μL、10mMdNTPs0.35μL、0.1μM引物2.0μ1、5UTaqDNA聚合酶0.15μL。最佳PCR扩增条件为:92℃预变性2min,1个循环;92℃变性1min,36℃退火1min,72℃延伸2min,共35个循环;72℃延伸5min,1个循环。

The RAPD reaction conditions on Eucalyptus wetarensis,such as PCR reaction mixture,PCR amplified program,and so on,were studied. The optimal reaction mixture(20μL total volume)contained 10μL genomic DNA,4.0μL ddH_2O,2.0μL 10×butter,15μL MgCl_2(25mM),035μL dNTPs(10mM),20μL primer and 015μL Taq DNA polymerase.Amplified program was as follows:preliminary denaturation(2min,92℃),followed by 35 cycles consisting of denaturation(1min,92℃),annealing(1min,36℃)and extension(2min,72℃),and a final extension(5min,72℃).