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糖原中核酸的污染及线性聚丙烯酰胺作为替代共沉淀剂的评估。

Nucleic acid contamination of glycogen used in nucleic acid precipitation and assessment of linear polyacrylamide as an alternative co-precipitant.

机构信息

Department of Biology, University of Waterloo, Waterloo, Ontario, Canada.

出版信息

Biotechniques. 2009 Dec;47(6):1019-22. doi: 10.2144/000113276.

Abstract

Molecular-grade glycogen is widely used to recover nanogram or picogram quantities of DNA and RNA across molecular biology applications in the life sciences. As a result, its purity is critical to obtain reliable results. Using agarose gel electrophoresis, we detected pg/microL (DNA) to ng/microL (RNA) concentrations of nucleic acid in two of the nine glycogen samples obtained from commercial suppliers. Denaturing gradient gel electrophoresis of 16S rRNA gene PCR-amplified products indicated that an additional two samples contained detectable contamination. We also tested a synthetic polymer co-precipitant, linear polyacrylamide (LPA); none of the four samples tested with LPA were detectably contaminated. The partial 16S rRNA gene sequence associated with the contaminated samples of the shellfish-derived glycogen was nearly identical to the sequence of Actinobacteria lwoffii, which has been isolated from mussels previously. By testing the recovery of low-nanogram amounts of DNA with multiple precipitants and simulated experimental conditions, we demonstrated that LPA was a preferable co-precipitant for sensitive protocols.

摘要

分子级糖原广泛应用于生命科学领域的分子生物学应用中,以回收纳克或皮克数量的 DNA 和 RNA。因此,其纯度对于获得可靠的结果至关重要。我们使用琼脂糖凝胶电泳检测了从商业供应商处获得的 9 种糖原样品中的两种,结果显示其 DNA 浓度为 pg/μL(DNA)至 ng/μL(RNA),16S rRNA 基因 PCR 扩增产物的变性梯度凝胶电泳表明,另外两种样品存在可检测的污染。我们还测试了一种合成聚合物共沉淀剂,即线性聚丙烯酰胺(LPA);用 LPA 测试的四个样品均未检出明显的污染。与贝类来源的糖原污染样品相关的 16S rRNA 基因部分序列与 Actinobacteria lwoffii 的序列几乎相同,该菌先前已从贻贝中分离出来。通过用多种沉淀剂和模拟实验条件测试回收低纳克数量的 DNA 的效果,我们证明 LPA 是敏感方案的首选共沉淀剂。

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