胡萝卜ISSR反应体系优化的研究

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。

Study on Optimization of ISSR Reaction System for Carrot

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.