正交设计优化东亚砂藓DDRT-PCR反应体系

详细信息

Optimization for DDRT-PCR System of Racomitrium japonicum Using Orthogonal Design

Department of Biology, College of Life Science and Engineering, Qiqihar University, Qiqihar, Heilongjiang 161006, China

摘要: 利用正交实验设计L₂₅(5⁶)对东亚砂藓(Racomitrium japonicum)DDRT-PCR反应体系的6因素(Mg²⁺、dNTP、锚定引物、随机引物、模板DNA、Taq酶)在5个水平上进行优化实验。结果筛选出各反应因素的最佳体系(20μL)为:Mg²⁺2.25mmol/L、dNTP0.4mmol/L、锚定引物1.0μmol/L、随机引物0.7μmol/L、模板DNA1.6μL、Taq酶2.5U。对东亚砂藓DDRT-PCR最佳反应体系进行梯度PCR引物退火温度筛选,得到的最佳退火温度为45.4℃。该优化体系的建立,为进一步进行东亚砂藓抗旱基因的筛选与克隆等一系列分子研究提供了重要参考依据。
- 正交设计 /
- 东亚砂藓 /
- DDRT-PCR
Abstract: In this paper,the orthogonal design was used to optimize DDRT-PCR amplification system on Racomitrium japonicum in five levels of six factors(Mg²⁺,dNTP,anchor primer,random primer,DNA template,Taq DNA polymerase) respectively.We established a most suitable DDRT-PCR system for R.japonicum,which contains Mg²⁺ 2.25 mmol/L,dNTP 0.4 mmol/L,anchor primer 1.0 μmol/L,random primer 0.7 μmol/L,DNA template 1.6 μL,and Taq DNA polymerase 2.5 U in 20 μL reaction system.At the last,the optimal annealing temperature for DDRT-PCR reaction was proposed by gradient PCR and it’s 45.4℃.The result provided an important reference for the selecting and cloning of drought-resistant genes of R.japonicum.
- Orthogonal design /
- Racomitrium japonicum /
- DDRT-PCR