牡丹ISSR-PCR反应体系正交优化设计

以“凤丹(”Paeonia ostii)基因组DNA为模板,采用正交试验设计方法,研究牡丹ISSR反应体系的影响因素,建立了适合牡丹的ISSR反应体系及程序。10 lμ反应体系为:1×反应缓冲液,2.5 mmo1/LMg2+,0.4 mmo1/LdNTPs,1.0 UTaq聚合酶,0.75μmol/L引物,10～20 ng模板DNA。反应程序为:94℃预变性3 min;94℃变性45 s,45～60(℃不同引物退火温度各异)复性45 s,72℃延伸90 s,35个循环;72℃延伸7 min;4℃保存。通过梯度退火试验,确定不同引物的退火温度。

Study on Optimization for ISSR Reaction System of Peony with Orthogonal Design

With Paeonia ostii genomic DNA as a template,the orthogonal design was used to optimize ISSR amplification system of Peony.A suitable ISSR reaction system was established,namely 10 μl reaction system containing 1×PCR buffer,2.5 mmol/L Mg2+,0.4 mmol/L dNTPs,1UTaq DNA polymerase,0.75 μmol/L primer,10~20 ng DNA templet.PCR reactions were pre-denaturing at 94 ℃ for 3 min,35 cycles of de-naturation at 94 ℃ for 45 s,annealing at 45~60 ℃ for 45 s and extension at 72 ℃ for 90 s,with a 7 min final extension at 72 ℃,and then saved at 4 ℃.The optimal annealing temperature for ISSR-PCR reaction was proposed by gradient PCR.Each primer has different annealing temperature.