白菜RAPD反应条件的优化

Optimalization of RAPD reaction system for Brassica campestris

以芜菁、大白菜及其杂交 1代为试材 ,系统分析了 Mg Cl2 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 2 0μL,Mg Cl2 浓度为 1 .5mmol/L ,d NTP浓度为 2 0 0μmol/L ,随机引物浓度为 0 .4μmol/L ,模板 DNA为 2 0 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℃ /1 0″预扩增 2循环 ;94℃ /1 0″→ 36℃ /1 5″→ 72℃ /70″扩增38循环 ;72℃

In this paper,the effects of concentration of MgCl 2,dNTP,random primers,template DNA,Taq DNA polymerase and different buffers on RAPDs were analyzed with turnip,Chinese cabbage and their hybrid F 1.An optimalized RAPD system with total volume of 20 μL including MgCl 2 at 1.5mmol/L,dNTP at 200 μmol/L,random primer at 0.4 μmol/L,20 ng template DNA,and 6.67 μmol/(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.