大白菜非变性聚丙烯酰胺凝胶电泳技术的优化

非变性聚丙烯酰胺凝胶电泳技术的检测效果受到凝胶聚合速度、片段迁移率等多种因素影响。以大白菜为供试材料,研究比较了凝胶聚合速度及PCR产物在不同浓度凝胶上的检测效果。不同浓度的凝胶是由2种〔40%(19∶1)和30%(29∶1)〕贮存液分别配制的4种浓度(5%,7%,9%,11%)。结果表明,在4～30℃,凝胶聚合速度随温度的升高而加快;APS保持不变,不同温度下TEMED∶APS合适配比也不同:4℃(冬季)时,TEMED/APS的合适配比是1∶10,25℃(春秋)、30℃(夏季),选择1∶20的配比为宜;2种不同贮存液配制的相同浓度凝胶检测效果不同,30%(29∶1)优于40%(19∶1),由前者配制7%的凝胶上DNA片段分离效果好,带型清晰,分辨率高。

The Optimization of Technique of Native Polyacrylamide Gel Electrophoresis (Native-PAGE)

Many factors including gel’s polymerization rate and fragments’ migration ratio affect detection effect of native polyacrylamide gel electrophoresis(Native-PAGE).The gel’s polymerization rate and the detection effect of PCR products by two kinds of gel stock solution(40% Acr-Bis(19∶1) and 30% Acr-Bis(29∶1) prepared in four concentrations(5%,7%,9%,11%) were studied in this paper.The results showed that the gel’s polymerization rate accelerated with increasing temperature in the range of 4-30 ℃,APS remains the same at different temperatures and TEMED∶APS suitable ratio was different: at 4 ℃(winter),the ratio was 1∶10,at 25 ℃ or 30 ℃(spring and Autumn)(summer),the ratio of 1∶20 was better.Detection of the effect was different in gel by two different stock solution prepared same gel concentrations;30% Acr-Bis(29∶1) is better than 40% Acr-Bis(19∶1),and DNA fragment separation revealed clear band type,high resolution in the gel that was prepared using 7% stock solution.