博白大果油茶ISSR-PCR反应体系的建立与优化

摘要

Taking Camellia gigantocarpa Hu et Huang as material, through the single factor design and orthogonal design L18 (35), the effects of major components on PCR reaction system, and the PCR amplification procedures were optimized. A stable, repeatable ISSR-PCR amplification reaction system for Camellia gigantocarpa was established. The optimal 20 μL reaction system' s parameters were as follows: 2.5 mmol/L Mg2+ , 20 ng template DNA, 1.75 U Taq DNA polymerase, 0. 25 mmol/L dNTPs, 0. 6 μmol/L primer. The optimal reaction procedure was shown as below: first pre-degeneration at 94℃ for 4 minutes, followed by 35 cycles at 94℃ for 30 seconds,annealing for 45 seconds at 50℃, and extending for 90 seconds at 72℃, and then terminated for 7 minutes at 72℃,finally stored at 4℃.%以博白大果油茶Camellia gigantocarpn Hu et Huang为材料,通过单因素实验设计和正交设计L18(35)对影响PCR反应体系的主要成分及PCR扩增程序进行优化.建立了稳定的、可重复的博白大果油茶ISSRPCR扩增反应体系.在20μL的反应体系中,Mg2+浓度为2.5 mmol/L,模板DNA用量为20 ng,Taq DNA聚合酶为1.75 U,dNTPs浓度为0.25 mmol/L,引物浓度为0.6 mm01/L.反应程序为:94℃预变性4 min,94℃变性30 s,50℃退火45 s,72℃延伸90 s,35个循环,72℃延伸7 min,4℃保存.