摘要
以早熟禾基因组DNA为模板,利用正交设计,对影响ISSR反应体系的主要因素(Mg2+、dNTP、Taq酶)从4个水平上进行筛选和优化,建立了适合早熟禾的最佳ISSR反应体系:25μl反应体系中1.25U Taq酶、1.5mmol/L Mg2+,10×PCR Buffer 2.5μl、0.4 mmol/L dNTP、1μl引物、1μl DNA模板。扩增程序为:94℃预变性3min;94℃变性30s,56.4℃退火30s,72℃延伸1min,循环35次;72℃延伸7min,4℃保存。在此反应体系下,可以得到重复性好、稳定且多态性高的条带。
Template DNA used for ISSR was extracted from Poa L. leaf tissue. The orthogonal design was applied to optimize ISSR amplification system at four levels of three factors （Taq DNA polymerase, Mgz ＋, dNTP） respectively. An optimal reaction system was established, 25ul reaction system contained 1.25U Taq DNA polymerase, 1.5 mmol · L^-1 Mg^2＋ ,10×PCR Buffer 2.5/11, 0.4mmol · L^-1 dNTP,1ul template DNA, 1ul primer. Amplification program included pre-denaturation at 94℃ for 7min; denaturation at 94℃ for 1 rain ; annealing at 56.4℃ for 30s; extending at 72℃ for 1min; after 35 cycles, the product was extended at 72℃ for 7min and kept at 4℃. Clear, steady and higher polymorphic bands were obtained through this optimized system.
出处
《中国草地学报》
CSCD
北大核心
2009年第5期107-111,共5页
Chinese Journal of Grassland
基金
甘肃省自然基金
农业行业专项(nyhyzx07-022)
科技支撑项目(2008BADB3B07)资助
