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液化低熔点琼脂糖凝胶直接消化DNA法在构建突变体中的应用

Application of Making Mutants by the Method of Direct Digestion DNA in Liquified Low Melting Agarose Gel
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摘要 用多级多聚酶链反应与液化低熔点琼脂糖凝胶中直接消化DNA相结合方法构建了8个突变体,经证实其重组DNA的阳性率为100%。从低熔点琼脂糖凝胶中切下目的DNA,在68℃完全熔化后用5倍体积的双蒸水稀释,该化合物即可直接用于酶解反应。混合物中凝胶比例以10g/L为宜。因而克服了凝胶中因目的DNA量少而纯化后回收率低无法进一步实验的弱点。 In this paper,the method of making mutants by multiple polymerase chain reaction (PCR) combined with direct digestion DNA in liquified low melting agarose gel was reported. We have made 8 mutants by the method described here. It was confirmed that the positive rate of recombination DNA was 100 percent. The slice of interesting DNA was taken out from the low melting agarose gel and was completely melted at 68℃. Then the melted slice was diluted with 5 volumes of distilled deionized water. The mixture could be then directly used for digesting DNA with appropriate enzyme. The suitable percentage of the agarose gel in mixture should be less then 1%. As a result, the shortcoming was overcome that the low yield of interesting DNA after purification can impossibly be used for further experiment.
出处 《同济医科大学学报》 CSCD 1997年第5期355-358,共4页 Acta Universitatis Medicinae Tongji
关键词 电泳 琼脂糖凝胶 重组 突变体 DNA electrophoresis agarose gel deoxyribonucleic acid recombinaiton mutation
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参考文献2

  • 1Chen H J,J Biol Chem,1997年,272卷,7003页 被引量:1
  • 2萨姆布鲁克 E F J,分子克隆实验指南(第2版),1996年,318,323页 被引量:1

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