A NEW METHOD FOR PURIFYING LAMBDA-DNA FROM PHAGE LYSATES
A NEW METHOD FOR PURIFYING LAMBDA-DNA FROM PHAGE LYSATES
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DOI：
10.1089/dna.1985.4.39
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发表时间：
1985-01-01
期刊：
影响因子：
--
通讯作者：
OLSON, MV
中科院分区：
文献类型：
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作者：
HELMS, C;GRAHAM, MY;OLSON, MV
一种制备少量 lambda 的新方法。开发了来自噬菌体裂解物的 DNA。该方案基于使用小型 DEAE 纤维素柱从粗裂解物中浓缩和纯化噬菌体颗粒。该色谱步骤给出了λ的绝对分离。细胞核酸中的 DNA 相对于粗裂解物中的主要可溶性蛋白质富集 20 倍，同时噬菌体浓度提高 10 倍。最终的脱蛋白和 λ 的浓缩。 DNA 通过常规沉淀步骤获得。 .lambda。通过这种方法产生的 DNA 是不可降解的、具有生物活性并且是限制性内切酶的优良底物。提供了从单个噬斑开始并使用该方法从大量 lambda 中获得纯化 DNA 的详细方案。克隆。
A new method for preparing small quantities of .lambda. DNA from phage lysates was developed. The protocol is based on the concentration and purification of bacteriophage particles from crude lysates using small DEAE-cellulose columns. This chromatographic step gives an absolute separation of the .lambda. DNA from the cellular nuclei acids and a 20-fold enrichment relative to the major soluble proteins in crude lysates, while effecting a 10-fold concentration of the phage. Final deproteinization and concentration of the .lambda. DNA is achieved by conventional precipitation steps. The .lambda. DNA produced by this method is nondegraded, biologically active and an excellent substrate for restriction enzymes. A detailed protocol is provided for starting with individual plaques and using the method to obtain purified DNA from large numbers of .lambda. clones.