甜菜ISSR-PCR反应体系的优化

Optimized Studies on Sugarbeet ISSR-PCR Reaction System

以甜菜基因组DNA为模板,研究了ISSR的主要影响因素,建立一套适宜于甜菜的ISSR优化反应体系及程序,并对该优化体系进行了验证。实验结果表明:①优化的ISSR扩增的反应体系为:20μL的反应体积中包括2.5mmol/LMgCl2,0.25mmol/LdNTPs,1.5UTaqDNA聚合酶,0.5μmo1/L引物,10×PCRBuffer2μL,100ngDNA模板。②优化的PCR扩增条件为:94℃预变性3min,然后94℃变性30s,45℃退火30s,72℃延伸1min,35个循环后,72℃延伸5min。③利用优化的反应体系仅用1条引物就能将10份甜菜材料区分开。

The main effect factors to sugarbeet ISSR were tested using DNA genome template, to build and identify the optimized ISSR reaction system and program which was suit to sugarbeet. The results showed that the optimized ISSR augmentation reaction system made up of MgCl2 2.5 mmol/L, dNTPs 0.25 retool/L, DNA polymerase 1.5 U Taq, primer 0.5μmol/L, Buffer 2 μL 10×PCR, DNA stencil 100 ng. The optimized condition was 3 rain pre-denaturization at 94℃, then 30s denaturalization 94℃, 30s annealing at 45℃, 1 min extension at 45℃, after 35 cycles, 5 min extension at 72℃. Ten sugarbeet varieties could be separated by one primer.