CAJ | 학술논문

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。
위건립삼목SRAP-PCR반응체계，이용L16（45）정교설계대영향삼목SRAP-PCR반응체계적Mg2＋、dNTPs、인물、Taq매화DNA농도5충인소적4개수평진행우화실험，결합정교직관분석화방차분석，대영향반응교대적Mg2＋、dNTPs화인물농도진행단인소실험，최종학정삼목SRAP-PCR최가적반응체계위：재20μL적PCR반응체계중，Mg2＋농도위2．25 mmol/L、dNTPs위0．15 mmol/L、인물농도위0．4μmol/L、Taq매위1．5μmol/min、모판DNA위60 ng，10×PCR Buffer 2μL，불족부분용쌍증수보충지20μL。 PCR반응정서적량보최괄퇴화온도제1보위35℃，제2보위53℃。이용상술반응체계진행삼목PCR확증，능득도청석、은정적조대。
In order to optimize SRAP-PCR reaction system for Chinese fir,the orthogonal design L16(45) was considered with different concentrations of Mg2+, 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 Mg2+, dNTPs,primer were rectified with single-factor design. Finally,the results showed that the best reaction system was the combination in 20μL reaction solution,containing of Mg2+ 2.25 mmol/L,dNTPs 0.15 mmol/L,primer 0.4 μmol/L,Taq DNA polymerase1.5μmol/min and DNA template 60 ng,2μL10×PCR Buffer and ddH2 O to 20μL. The first optimal an-nealing 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.