北沙柳SSR-PCR反应体系优化及引物筛选
首发时间:2012-11-15
摘要:以TIANGEN试剂盒法提取北沙柳嫩叶的DNA为模板,利用柳属和杨属的SSR引物,建立了北沙柳SSR-PCR反应体系,对反应体系中影响聚合酶链式反应(PCR)的因素进行了优化。结果表明,在25μl的PCR反应体系中,DNA模板用量为40ng,TaqDNA聚合酶0.05U/μl,10×PCR buffer(含Mg2+)2.5mM,dNTP 0.25mM,SSR引物0.5μM,加ddH2O补足至25μl时结果最好。利用该体系初步筛选出30对适合北沙柳SSR扩增的引物,并采用优化筛选出的12对引物对9个产地的270个北沙柳无性系进行PCR扩增,多态位点百分率达到90.4%,表明柳属和杨属的SSR引物可以在北沙柳上应用。
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Optimization of the SSR-PCR System and Selection of Primers in Salix psammophila
Abstract:Using TIANGEN extraction Salix psammophila leaves DNA as a template, use of Salix and Populus SSR primer, established the Salix psammophila SSR-PCR reaction system, the reaction system of PCR factors were optimized.The result indicated that the optimized SSR reaction system for the plant was 40 ng template DNA,0.05 U/μl Taq DNA polymerase,2.5 mmol 10×PCR buffer(Mg2+),0.25 mmol dNTP,and 0.5μmol primer,In 25μl reaction volumes.SSR-PCR reaction system was determined on a total volume 25μl system,12 Salix psammophila SSR primers were selected.Clones from 9 original areas were amplified by 12 primers.with a percentage of 90.4 in total, Salix and Populus SSR primers can be used in the Salix psammophila.
Keywords: Salix psammophila SSR-PCR optimization of reaction system primer screening
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