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通过硫酸铵沉淀法纯化噬菌体λgt11 DNA,用于克隆cDNA插入片段的限制性分析。

Purification by ammonium sulfate precipitation of bacteriophage lambda gt11 DNA for restriction analysis of cloned cDNA inserts.

作者信息

Ziai M R, Giordano A, Armandola E, Ferrone S

机构信息

Department of Microbiology and Immunology, New York Medical College, Valhalla 10595.

出版信息

Anal Biochem. 1988 May 15;171(1):192-6. doi: 10.1016/0003-2697(88)90141-8.

Abstract

A rapid and efficient method to purify lambda gt11 DNA is described. This technique involves precipitation of intact bacteriophage particles with ammonium sulfate, followed by phage lysis with sodium dodecyl sulfate, proteinase K, and alkaline treatment. The quality of DNA for subsequent restriction analysis, infectivity, subcloning, and radiolabeling is comparable to that isolated by cesium chloride banding or ion exchange chromatography. The yield of the phage DNA is, however, two to eight times higher than that obtained by other conventional methods of lambda gt11 purification. Furthermore the time required to process the bacteriophage lysate is approximately 2 h and therefore more rapid than other currently used methods.

摘要

本文描述了一种快速有效的纯化λgt11 DNA的方法。该技术包括用硫酸铵沉淀完整的噬菌体颗粒,然后用十二烷基硫酸钠、蛋白酶K进行噬菌体裂解以及碱性处理。用于后续限制性分析、感染性、亚克隆和放射性标记的DNA质量与通过氯化铯密度梯度离心或离子交换色谱法分离得到的DNA质量相当。然而,噬菌体DNA的产量比通过其他传统的λgt11纯化方法获得的产量高两到八倍。此外,处理噬菌体裂解物所需的时间约为2小时,因此比目前使用的其他方法更快。

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