拟南芥SSR检测体系的优化

以拟南芥(Arabidopsis thaliana)幼苗为实验材料,采用单因素筛选法及L16(45)正交实验方法,对简单序列重复(SSR)技术中聚合酶链式反应(PCR)组分、扩增程序、电泳检测等环节进行优化。优化25μl反应体系为:1×PCR Buffer、20ng模板DNA、1.5mmol.L-1Mg2+、0.3μmol·L-1引物、150μmol·L-1dNTPs和1.0U Taq DNA聚合酶。扩增程序为:94℃预变性5min,94℃变性30s,57℃退火30s,72℃延伸45s,共30个循环,72℃延伸10min。用非变性聚丙烯酰胺凝胶(EB染色)电泳检测并取得较好效果。利用该体系进行扩增,所得谱带清晰、稳定、非特异性带少。

Optimization of SSR deletion system for Arabidopsis thaliana

Taking Arabidopsis thaliana as test material and by the methods of single factor selection and L_(16)(4~5)orthogonal design,the amplification component,program,and electrophoresis detection in polymerase chain reaction(PCR)in simple sequence repeat(SSR)-detection system were optimized.The optimized 25 μl reaction system contained 1×PCR Buffer,20 ng DNA template,1.5 mmol·L~(-1) of Mg~(2+),0.3 μmol·L~(-1) of primers,150 μmol·L~(-1) of dNTPs,and 1.0 U of Taq DNA polymerase.The PCR amplification procedures consisted of an initial denaturing for 5 min at 94 ℃,followed by 30 cycles of denaturation for 30 s at 94℃,annealing for 30 s at 57℃,and an extension for 45 s at 72℃,with an additional extension period of 10 min at 72 ℃.By using non-denaturing polyacrylamide gel electrophoresis with ethidium bromide(EB)staining,the amplification products bands of SSR were easy to be identified.In the SSR system,amplification bands were clear and stable,and there were fewer non-target bands.