摘要
以河鲈为研究材料,对河鲈RAPD-PCR的反应体系进行优化和适用引物的筛选,采用正交设计方法,选用L17(54)正交表,以河鲈基因组DNA为模板,对影响PCR反应较大的4个因素:Taq酶、Mg2+、dNTPs、引物在5个水平上进行优化试验,得到河鲈最优RAPD-PCR反应体系:25μL反应体系中含有30 ng DNA模板1.5μL,10μmol/L引物0.7μL,10×buffer 2.5μL,25μmol/L dNTPs 0.4μL,5 U Taq酶0.2μL,2.5 mmol/L MgCl23.0μL;利用优化体系从50条引物中筛选得到10条适用引物,并确定了引物的最佳退火温度。
In order to get the best reaction system and suitable primerof Perca fluviatilis,usingPerca fluviatilisgenomic DNA as templates,by L17（54）orthogonal experiment table,four major factors（Taqenzyme,Mg2＋,dNTP and primer）that greatly affected the results of RAPD were chosen and design to seek optimal reaction condition.For a 25 μL reaction system contain,the optimal compositions included 30 ng DNA template 1.5 μL,10 μmol/L primer0.7 μL,10×buffer2.5 μL,5 μmol/L dNTPs 0.4 μL,5U Taqpolymerase 0.2 μL,2.5 mmol/L MgCl2 3.0 μL;useing the optimal system,10 random primers were selected from 50 primers,and determine the optimal annealing temperature.
出处
《生物技术通报》
CAS
CSCD
北大核心
2013年第9期114-118,共5页
Biotechnology Bulletin
基金
石河子大学动植物育种专项(gxjs2011-yz01)
关键词
河鲈
RAPD-PCR
条件优化
适用引物
退火温度
Perca fluviatilis RAPD-PCR Condition optimization Suitable primers Annealing temperature
