摘要
以改良CTAB法提取胡萝卜基因组DNA作为ISSR-PCR模板,通过单因素试验建立了一套适合胡萝卜ISSR-PCR的优化的反应体系,即25μL反应液中含10×buffer2.5μL,2.0mmol.L-1Mg2+,200μmol·L-1dNTPs,Taq酶1.5U,引物0.5μmol·L-1,DNA模板20ng。PCR扩增程序为:94℃预变性4min,94℃变性40s,48～58℃退火45s,72℃延伸2min,进行35个循环,72℃延伸8min,在16℃保存。应用该优化反应体系筛选出了24条有效引物。
The improved CTAB method was used to extract genomic DNA of Daucus carota L. var. sativa.,which were used as the template for ISSR-PCR. A optimal ISSR-PCR reaction system suitable to Daucus carota L. var. sativa DC. was established via single factor experiments. The optimal ISSR-PCR system was that 25 μL reaction solution contained 2.5 μL 10×PCR buffer,2.5—3.0 mmol·L-1 Mg2＋,200μmol·L-1 dNTPs,1.5U Taq polymerase,0.5 μmol·L-1 primers,20ng DNA template.The optimized amplification program was that pre-denaturing at 94 ℃ for 4min,then denaturing at 94 ℃ for 40 s,primer annealing at 48 ℃—58 ℃ for 45 s,extension at 72 ℃ for 2 min,for 35 cycles,extension at 72 ℃ for 8min. The products were stored at 16 ℃. 24 effective primers were screened out.
出处
《热带生物学报》
2010年第4期301-306,313,共7页
Journal of Tropical Biology
基金
海南省耐盐作物生物技术重点实验室开放基金资助项目(shkfjj0515)
关键词
胡萝卜
ISSR
优化
Daucus carota L. var. sativa DC.
ISSR
optimization
