杉木SRAP-PCR体系优化  

Optimization of Chinese fir SRAP-PCR system

作　　者：王新民[1] 郑仁华[2] 陈金慧[1] 赵亚琦[1] 徐阳[1] 王颖[1] 施季森[1] 

机构地区：[1]南京林业大学,林木遗传与生物技术省部共建教育部重点实验室,江苏南京210037 [2]福建省林业科学研究院,福建福州350012

出　　处：《南京林业大学学报（自然科学版）》2014年第1期15-20,共6页Journal of Nanjing Forestry University：Natural Sciences Edition

基　　金：国家林业公益性行业科研专项项目(201004049);国家自然科学基金重点项目(30930077);江苏高校优势学科建设工程资助项目(PAPD)

摘　　要：为建立杉木SRAP-PCR反应体系,利用L16(45)正交设计对影响杉木SRAP-PCR反应体系的Mg2+、dNTPs、引物、Taq酶和DNA浓度5种因素的4个水平进行优化实验,结合正交直观分析和方差分析,对影响反应较大的Mg2+、dNTPs和引物浓度进行单因素实验,最终确定杉木SRAP-PCR最佳的反应体系为:在20μL的PCR反应体系中,Mg2+浓度为2.25 mmol/L、dNTPs为0.15 mmol/L、引物浓度为0.4μmol/L、Taq酶为1.5μmol/min、模板DNA为60 ng,10×PCR Buffer 2μL,不足部分用双蒸水补充至20μL。PCR反应程序的两步最适退火温度第1步为35℃,第2步为53℃。利用上述反应体系进行杉木PCR扩增,能得到清晰、稳定的条带。In order to optimize SRAP-PCR reaction system for Chinese fir,the orthogonal design L16（4^5） was considered with different concentrations of Mg^2＋, dNTPs, primer, Taq DNA polymerase and DNA template in SRAP-PCR reaction system for Chinese fir. The results of PCR were evaluated by visual and variance analysis ,then the concentrations of Mg^2＋, dNTPs,primer were rectified with single-factor design. Finally, the results showed that the best reaction system was the combination in 20 μL reaction solution, containing of Mg^2＋ 2.25 mmol/L, dNTPs 0.15 mmol/L, primer 0.4 μmol/L, Taq DNA polymerasel.5 p.mol/min and DNA template 60 ng,2 μL10xPCR Buffer and ddH20 to 20 μL. The fimt optimal an- healing temperature was 35 ℃ ,and the second step was 53 ℃. The optimized reaction system of Chinese fir described a- bove can get clear and stable bands.

关 键 词：杉木 SRAP-PCR 体系优化 

分 类 号：S722[农业科学—林木遗传育种] Q81[农业科学—林学]