| 摘要: |
| 以芜菁、大白菜及其杂交1代为试材 ,系统分析了MgCl2 浓度、d NTP浓度、随机引物浓度、模板DNA用量、Taq DNA聚合酶浓度以及不同来源扩增缓冲液对 RAPD反应的影响 ,建立了一套稳定的RAPD反应体系。该体系反应体积为 20μL,MgCl2 浓度为 1.5mmol/L ,dNTP浓度为 200μmol/L ,随机引物浓度为0.4μmol/L ,模板DNA为20 ng,TaqDNA聚合酶为 6.67μmol/(L·min)。在 8种热循环条件下 ,该体系具有稳定的重现性 ,其扩增程序为 :94℃/1′→36℃/2 0″→72℃/10″预扩增 2循环;94℃/10″→36℃/15″→72℃/70″扩增38循环;72℃/4′延伸效果最好 |
| 关键词: 大白菜,RAPD,反应条件 |
| DOI： |
| 分类号:S634.1 |
| 基金项目:高等学校博士学科专项科研基金资助项目(960701)，北京市蔬菜种质改良重点实验室资助项目 |
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| Optimalization of RAPD reaction system for Brassica campestris |
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ZHANG Lu gang 1 2 WANG Ming 2 CHEN Hang 3 LIU Ling 3 Samuel S.M.Sun 4
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| Abstract: |
| In this paper, the effects of concentration of MgCl2, dNTP, random primers, template DNA, Taq DNA polymerase and different buffers on RAPDs were analyzed with turnip,Chinese cabbage and their hybrid F1.An optimalized RAPD system with total volume of 20μL including MgCl2 at 1.5mmol/L, dNTP at 200μmol/L,random primer at 0.4μmol/L, 20 ng template DNA, and 6.67μmol/(L·min) (unit) of Taq DNA polymerase was set up.This system showed stable repetition at 8 temperature cycle condition, in which one program with pre-amplification 94℃/1′→36℃/20″→72℃/10″for 2 cycles; amplification 94℃/10″→36℃/15″→72℃/70″for 38 cycles; and extension at 72℃for 4 min presented a better result. |
| Key words: Chinese cabbage,RAPD,amplification condition |
