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.
出处
《南京林业大学学报(自然科学版)》
CAS
CSCD
北大核心
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)