Extraction of Superior-Quality Plasmid DNA by a Combination of Modified Alkaline Lysis and Silica Matrix

Ramakrishna, Lakshmi; Baskar, Vijay; Ranga, Udaykumar

doi:10.1006/abio.1999.4125

Cited by 27 publications

(21 citation statements)

References 7 publications

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“…A wide variety of protocols have been developed using native unmodified silica resin (511) or its derivatives, such as diatomaceous earth (12) or pumice (13) for preferential isolation of nucleic acids. Although the quality and quantity of the nucleic acids extracted using the crude silica are superior to the conventional techniques, they are certainly not suitable for a range of applications, such as in vitro transcription/translation, microinjection, and immunization (8). When purity of the nucleic acids is an important consideration, commercial nucleic acid extraction columns have no substitute.…”

Section: Introductionmentioning

confidence: 99%

Regeneration of Commercial Nucleic Acid Extraction Columns without the Risk of Carryover Contamination

et al. 2007

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Nucleic acid extraction is a basic requirement in a molecular biology laboratory. In terms of purity and yield, commercial nucleic acid extraction columns are superior; however, they are expensive. We report here an efficient strategy to regenerate diverse commercial columns for several rounds without altering the binding capacity of the columns or changing the properties of the nucleic acids purified. Plasmids purified with regenerated columns were functionally identical in super-coiled nature, restriction analysis, expression of the encoded reporter genes, or amplification of the viral RNA in real-time PCR. To ensure that the regenerated columns were free of the residual DNA, we used two different plasmids with different drug-resistance markers. By colony plating and PCR amplification of the encoded genes, we show that the regeneration process is absolute. Using radiolabeled DNA, we demonstrate that DNA exposed to the regeneration reagent is fragmented to molecular weight below 36 bp. Our data collectively prove regeneration of the commercial columns without the concern of carryover contamination. A procedure to permit safe and efficient regeneration of the commercial columns is not only of great advantage to extend the lifetime of these columns but also makes them commercially more affordable, especially in a resource-poor setting.

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Section: Introductionmentioning

confidence: 99%

Regeneration of Commercial Nucleic Acid Extraction Columns without the Risk of Carryover Contamination

et al. 2007

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“…size-exclusion [6][7][8], reversed-phase [9,10], hydrophobic interaction [11][12][13], hydroxyapatite [14][15][16], silica [17][18][19] and triple-helix affinity [20,21]. Anion-exchange remains the most popular chromatography technique as it offers the advantages of rapid separation, no solvent requirement, sanitisation with sodium hydroxide and a wide selection of industrial media.…”

Section: Introductionmentioning

confidence: 99%

Purification of pharmaceutical-grade plasmid DNA by anion-exchange chromatography in an RNase-free process

Eon‐Duval

Burke

2004

Journal of Chromatography B

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“…Due to the ability of DNA to bind to silica in the presence of chaotropic salt (Boom et al, 1990), it has been widely used in DNA isolation, being a very efficient, inexpensive and rapid means to isolate DNA from several sources (Carter andMilton, 1993: Hoss andPaabo, 1993;Lakshmi et al, 1999;Dederich et al, 2002;Li and Sheen, 2010).…”

Section: Discussionmentioning

confidence: 99%

A simple, fast, and inexpensive CTAB-PVP-silica based method for genomic DNA isolation from single, small insect larvae and pupae

Huanca‐Mamani¹,

Rivera-Cabello²,

Maita-Maita³

2015

Genet. Mol. Res.

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ABSTRACT. In this study, we report a modified CTAB-PVP method combined with silicon dioxide (silica) treatment for the extraction of high quality genomic DNA from a single larva or pupa. This method efficiently obtains DNA from small specimens, which is difficult and challenging because of the small amount of starting tissue. Maceration with liquid nitrogen, phenol treatment, and the ethanol precipitation step are eliminated using this methodology. The A 260 /A 280 absorbance ratios of the isolated DNA were approximately 1.8, suggesting that the DNA is pure and can be used for further molecular analysis. The quality of the isolated DNA permits molecular applications and represents a fast, cheap, and effective alternative method for laboratories with low budgets.

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Extraction of Superior-Quality Plasmid DNA by a Combination of Modified Alkaline Lysis and Silica Matrix

Cited by 27 publications

References 7 publications

Regeneration of Commercial Nucleic Acid Extraction Columns without the Risk of Carryover Contamination

Regeneration of Commercial Nucleic Acid Extraction Columns without the Risk of Carryover Contamination

Purification of pharmaceutical-grade plasmid DNA by anion-exchange chromatography in an RNase-free process

A simple, fast, and inexpensive CTAB-PVP-silica based method for genomic DNA isolation from single, small insect larvae and pupae

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