摘要
为了对影响银杏戗ngkobiloba简单序列重复区间扩增-聚合酶链式反应（ISSR-PCR）扩增反应体系的因素进行优化，采用[SSR-PCR扩增技术和UVP凝胶电泳成像技术对模扳DNA浓度、Taq酶用量、引物用量、dNTP的用量以及退火温度等因素进行筛选和优化筛选优化后的反应条件：Taq酶0．3μg，2μL10×Buffer（含15mmol·L^-1 MgCl2），模板DNA40ng，dNTP0．2mmol·L^-1，引物0．5μmol·L^-1，Mg^2＋，5nmol·L^-1。PCR扩增程序：94℃变性5min，然后进行38个循环：94℃变性30S，48～53℃（根据引物而定）退火30s，72℃延伸1min；最后72℃延伸10min，4℃终止反应。上述反应条件可广泛应用于银杏的遗传多样性分析、遗传育种和转基因等方面的研究。
To optimize the inter simple sequence repeat （ISSR） reaction conditions with Ginkgo biloba, the concentrations of template DNA, Taq DNA polymerase, primers, deoxyribonucleotide triphosphate （dNTP） , along with annealing temperature and their influence on polymerase chain reaction （PCR） amplification were studied. The sample leaves were collected in 97 old ginkgo trees from more than 20 provinces in April and May, 2006. Results showed that the optimum reaction conditions for ISSR-PCR of ginkgo were： 0. 3μg Taq DNA, 2 fl.L 10 x Taq buffer （including 15 mmol .L-·MgCl2）, 40 ng template DNA, 0.2 mmol· L^-1 dNTP, 0. 5μmol· L^-1 primer, and 1.5 mmol · L^-7 Mg^2＋. with a total volume of 20μL. Amplification consisted of one cycle with initial denaturation at 94℃ for 5 min, followed by 38 cycles of 30 s at 94 ℃, 30 s annealing at 48 -53 ℃ （depending on the primer used） , 1 min at 72 ℃, and a final 10 rain extension at 72℃. Afterward, the samples were maintained at 4 ℃. [ Ch, 6 fig. 1 tab. 19 ref.]
出处
《浙江林学院学报》
CSCD
北大核心
2008年第2期186-190,共5页
Journal of Zhejiang Forestry College
基金
"十一五"国家科技支撑项目(2006BAD18B0301)
