| 引用本文: | 韦荣昌, 李 锋, 黄夕洋, 蒋水元, 蒋向军, 戴 俊, 李志刚.多倍体罗汉果PCR-RFLP参数的优化与应用[J].广西植物,2009,(6):889-893.[点击复制] |
| WEI Rong-Chang, LI Feng, HUANG Xi-Yang, JIANG Shui-Yuan,
JIANG Xiang-Jun, DAI Jun, LI Zhi-Gang.Optimization and preliminary study on the PCR-RFLP parameter of polyploidy Siraitia grosvenorii[J].Guihaia,2009,(6):889-893.[点击复制] |
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| 多倍体罗汉果PCR-RFLP参数的优化与应用 |
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韦荣昌1,2, 李 锋2, 黄夕洋2*, 蒋水元2, 蒋向军3, 戴 俊4, 李志刚1
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1.广西大学, 南宁 530004;2. 广西壮族自治区
中国科学院 广西植物研究所, 广西 桂林 541006;3.桂林亦元生
现代生物技术有限公司, 广西 桂林541004;4.广西师范大学 生命科学学院, 广西 桂林 541004
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| 摘要: |
| 以多倍体罗汉果DNA为材料,采用L16(45)正交组合试验和单因素梯度试验,研究Mg2+、dNTP、引物、Taq DNA聚合酶、模板DNA浓度和退火温度、循环次数等对PCR扩增结果以及内切酶量、酶切时间对酶切反应的影响。结果表明,多倍体罗汉果RFLP最优PCR反应体系和扩增参数为:在25 μL扩增反应体系中,10×Buffer 2.5 μL,MgCl2 1.5 mmol/L,dNTP 0.2 mmol/L,引物0.1 μmol/L,Taq DNA聚合酶2.0 U,模板DNA 60 ng; 退火温度为56 ℃,循环次数为35次。酶切反应体系:内切酶10×Buffer 2.0 μL,内切酶5.0 U,PCR产物15 μL,超纯水补至20 μL; 酶切时间2 h。 |
| 关键词: 多倍体罗汉果 PCR-RFLP 酶切反应 优化与应用 |
| DOI： |
| 分类号:Q943 |
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| Optimization and preliminary study on the PCR-RFLP parameter of polyploidy Siraitia grosvenorii |
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WEI Rong-Chang1,2, LI Feng2, HUANG Xi-Yang2*, JIANG Shui-Yuan2,
JIANG Xiang-Jun3, DAI Jun4, LI Zhi-Gang1
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1.Guangxi University, Nanning 530004, China;2.Guangxi Institute of Botany, Guangxi Zhuang Autonomous
Region and Chinese Academy of Sciences, Guilin 541006, China;3.Guilin Sunnylife Modern Bio-
Tech INC., Guilin 541004, China;4.Guangxi Normal University, Guilin 541004, China
1.Guangxi University, Nanning 530004, China; 2.Guangxi Institute of Botany, Guangxi Zhuang Autonomous
Region and Chinese Academy of Sciences, Guilin 541006, China; 3.Guilin Sunnylife Modern Bio-
Tech INC., Guilin 541004, China; 4.Guangxi Normal University, Guilin 541004, China
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| Abstract: |
| In this paper,the DNA of polyploidy Siraitia grosvenorii was regarded as the research object,several important factors including MgCl2,dNTP,primer,Taq DNA polymerase,template DNA,annealing temperature,amplification and enzyme quantity,digestion reaction time were optimized by L16(45)orthogonal design experiment and single factor experiment in order to investigate the effects on the results of PCR amplification and digestion reaction. The test results showed that a total volume of 25 μL PCR reaction system contained 10×Buffer 2.5 μL,MgCl2 1.5 mmol/L,dNTP 0.2 mmol/L,general service primer 0.1 μmol/L,Taq DNA polymerase 2.0 U,template DNA 60 ng,and annealing temperature was 56 ℃,amplification for 35 cycles was optimizational PCR amplification reaction of RFLP of polyploidy S.grosvenorii. The total volume of 20 μL digestion reaction system contained digestion enzyme 10×Buffer 2.0 μL,digestion enzyme 5.0 U,PCR product 15 μL,digestion reaction time 2 h. |
| Key words: polyploidy Siraitia grosvenorii PCR-RFLP digestion reaction optimization and application |
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