不同酶切体系对胶回收纯化DNA浓度的影响  

Effects of Different Enzyme Systems on Concentration of DNA Purified by Gel

作　　者：徐珍[1] 张玉芹[1] 聂永莉[1,2] 

机构地区：[1]武汉科技大学医学院细胞与分子生物学研究所,430065 [2]湖北省丹江口汉江集团汉江医院心内科,442700

出　　处：《医学研究杂志》2010年第9期54-57,共4页Journal of Medical Research

基　　金：湖北省自然科学基金(2004ABA152);湖北省教育厅重点项目(D20081108)

摘　　要：目的通过建立不同的酶切体系,找寻酶切效果好、胶回收试剂盒纯化DNA得率高的实验设计。方法使用XhoⅠ单酶切重组质粒pcDNA3-P2X4,酶切体系的体积分别为:150μl、160μl、170μl、180μl;酶切体系组成为:dddH2O(补足体积)、BufferR(应用浓度是酶切体积的10%)、质粒DNA(无论所得质粒浓度如何,琼脂糖凝胶电泳胶回收的DNA上样量统一为23μg),XhoⅠ10μl(6μl、4μl两次加入);反应温度为:37℃;酶切的时间为2～3h。结果酶切体系越大,所需要的酶切时间越短、酶切效果越好、酶切条带越接近Marker标准条带(通过电泳拍照观察所得),但是胶回收纯化后DNA的浓度却越低:150μl体系DNA的浓度=308.96±8.71μg/ml,160μl体系DNA的浓度=286.62±8.37μg/ml,170μl体系DNA的浓度=245.80±15.64μg/ml,180μl体系DNA的浓度=198.00±16.54μg/ml(方差分析,P<0.05,n=5)。结论将酶切的体系放大,杂质将相对稀释,不需增加酶的用量就能取得较好的酶切效果;提取质粒的浓度和纯度也决定着酶切效果。Objective To find good digestion results as well as the high rate of purification of DNA gel extraction kit through establishing different enzyme systems. Methods Single - enzyme（Xho I ） digestion recombinant plasmid pcDNA3 - P2X4 was established by the volume of enzyme system of 150μl, 160μl, 170μl, 180μl respectively. Composition of the digest system was dddH2 0 （ fill volume）, Buffer R （ application of the concentration of enzyme volume 10% ）, plasmid DNA （ no matter what the concentration was, sample volume on the unification of DNA gel recovery was always 23μg） ,Xho I 10μl （5μl,4μl, adding seperatly）. Reaction temperature was 37℃ and digestion time was 2 - 3h. Results The the greater digestion system was that the shorter the time required for digestion, the better the digestion was and the closer the digestion band was to the standard Marker bands （photographed by electrophoresis observations）. However, the concentration of gel purified DNA was low as following： DNA concentration of 150μl system = 308.96 ± 8.71μg/ml, DNA concentration of 160μl system = 285.62 ± 8.37μg/ml, DNA concentration of 170μl system = 245.80 ± 15. 64μg/ml, DNA concentration of 180μl system= 198.00 ±16.54μg/ml （One-Way ANOVA, P〈0.05, n=5）. Conclusion Enlarging the digestion system, would lead to impurities be relatively diluted and would be able to achieve better result without increasing the amount of enzyme. The concentration and purity of extracted plasmid also determined the digestion results.

关 键 词：琼脂糖凝胶电泳 胶回收 单酶切 DNA纯化 

分 类 号：R714.244[医药卫生—妇产科学]