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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℃ /4′延伸效果最好
Using Chinese turnip, Chinese cabbage and their first generation hybrids as materials, the effects of MgCl 2 concentration, d NTP concentration, random primer concentration, template DNA concentration, Taq DNA polymerase concentration and different sources of amplification buffer on RAPD reaction were systematically analyzed , Established a stable RAPD reaction system. The reaction volume was 20 μL, the concentration of MgCl 2 was 1.5 mmol / L, the concentration of dNTP was 200 μmol / L, the concentration of random primer was 0.4 μmol / L, the template DNA was 20 ng and the concentration of Taq DNA polymerase was 6.67 μmol / (L · min). Under eight kinds of thermal cycling conditions, the system has a stable reproducibility. The amplification program is as follows: pre-amplification at 94 ° C / 1 ’→ 36 ° C / 20’ → 72 ° C / 100 ’for 2 cycles; 94 ° C / 1 0 “→ 36 ℃ / 1 5” → 72 ℃ / 70 "for 38 cycles; the best extension effect at 72 ℃ / 4 ’