A One-Step Miniprep for the Isolation of Plasmid DNA and Lambda Phage Particles

Lezin, George; Kosaka, Yasuhiro; Yost, H. Joseph; Kuehn, Michael R.; Brunelli, Luca

doi:10.1371/journal.pone.0023457

Cited by 25 publications

(26 citation statements)

References 33 publications

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“…Our current experience in DNA extraction and purification suggests that sample preparation in the liquid state is the most time and cost‐efficient approach. In particular, changing water properties including solutes solubility by the addition of salts, detergents, and organic solvents could replace expensive solid mass exchange resins with lengthy processing times . Consistent with this notion, we studied whether PCR products could be precipitated from iPrOH solutions of LiCl, a salt highly soluble in alcohol, simultaneously retaining 100‐200 bp PCR byproducts in the solutions.…”

Section: Resultsmentioning

confidence: 94%

“…The pattern of native plasmid DNA bands, formed in electrophoresis gels, partially depends on the DNA extraction method's capacity to inhibit intracellular endonucleases and topoisomerases. Unlike alkali extraction, NID buffers lacking inhibitors allow the extraction of sensitive lambda particles and the use of NID crude extracts for downstream applications . Thus, a difference in gel banding plasmid DNA extracted by alkali and NID is expected as it is shown in Figure A.…”

Section: Resultsmentioning

confidence: 99%

“…Alkali plasmid DNA extraction was done as originally described by Birnboim and Doly. 14 NID Plasmid DNA extraction was done as previously described, 15…”

Section: Dna Plasmid Isolationmentioning

confidence: 99%

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Live‐cell PCR and one‐step purification streamline DNA engineering

Lyozin

Brunelli

2020

FASEB j.

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In vivo DNA engineering such as recombineering (recombination‐mediated genetic engineering) and DNA gap repair typically involve growing Escherichia coli (E coli) containing plasmids, followed by plasmid DNA extraction and purification prior to downstream PCR‐mediated DNA modifications and DNA sequencing. We previously demonstrated that crude cell lysates could be used for some limited downstream DNA applications. Here, we show how live E coli cell PCR and one‐step LiCl‐isopropanol purification can streamline DNA engineering. In DNA gap repair, live‐cell PCR allowed the convenient elimination of clones containing background plasmids prior to DNA sequencing. Live‐cell PCR also enabled the generation of specific DNA sequences for DNA engineering up to 11 kilo base pairs in length and with up to 80 base pair terminal non‐homology. Using gel electrophoresis and DNA melting curve analysis, we showed that LiCl‐isopropanol DNA precipitation removed primers and small, nonspecific PCR products from live‐cell PCR products in only ~10‐minutes. DNA sequencing of purified products yielded Phred quality scores values of ~55%. These data indicate that live‐cell PCR and LiCl‐isopropanol DNA precipitation are ideal to prepare DNA for sequencing and other downstream DNA applications, and might therefore accelerate high‐throughput DNA engineering pipelines.

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Section: Resultsmentioning

confidence: 94%

Section: Resultsmentioning

confidence: 99%

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Live‐cell PCR and one‐step purification streamline DNA engineering

Lyozin

Brunelli

2020

FASEB j.

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“…Using "area" and "label peaks" commands, the percentage of the area of each band can be calculated. This technique has been employed to conduct image analysis in several studies [10,[27][28][29].…”

Section: Agarose Gel Electrophoresis Analysismentioning

confidence: 99%

“…Lezin et al [10], developing a one-step approach for isolate pDNA, found that non-ionic detergent (NID) plasmid DNA performs better than alkaline treatment in many downstream applications. Bag et al [11] developed an improved method for the extraction of community DNA from different environmental and human origin samples by a combination of physical, chemical, and mechanical lysis methods for proper lysis of microbial DNA.…”

Section: Introductionmentioning

confidence: 99%

Efficient Disruption of Escherichia coli for Plasmid DNA Recovery in a Bead Mill

et al. 2017

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Featured Application: In general, the alkaline lysis is more acceptable than the mechanical lysis for pDNA recovery due to the risk of product degradation when the latter is used. In this work, the experimental design allowed us to explore mechanical disruption conditions under which such an effect was minimized. The application of this technique is important for novel pDNA-vaccine process development, since bead milling is one of the most preferable mechanical cell lysis methods on an industrial scale due to its scalability, ease of operation, controllability, and ability to load concentrated cell slurry. Abstract:The release kinetics of pDNA in a bead mill was studied. Samples taken during the process were analyzed to determine total pDNA (pDNA(t)) and supercoiled pDNA (pDNA(sc)) concentration. In order to identify important variables of the process and to develop an empirical model for optimal pDNA(t) and pDNA(sc) release, a two level 2 3 factorial design was used with variables: mill frequency, cell concentration, and bead size. The results were analyzed by response surface methodology. The optimized conditions for pDNA(t) yield 13.26 mg/g dcw (93.41% recovery), with a mill frequency of 30 Hz, a bead size of 0.10-0.25 mm, and a cell concentration of 20 g wcw/L. However, the optimized conditions for pDNA(sc) yield 7.65 mg/g dcw (92.05% recovery), with a mill frequency of 15 Hz, a bead size of 0.10-0.25 mm, and a cell concentration of 10 g wcw/L. Cell disruption in a bead mill was proved efficient for the release of pDNA(t) and pDNA(sc) compared to the alkaline treatment. The results obtained suggest a compromise between pDNA(sc) purity and recuperation in the process development.

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Direct Isolation of Seamless Mutant Bacterial Artificial Chromosomes

Lyozin

Kosaka

Bhattacharje

et al. 2017

CP Molecular Biology

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Seamless (i.e., without unwanted DNA sequences) mutant bacterial artificial chromosomes (BACs) generated via recombination-mediated genetic engineering (recombineering) are better suited to study gene function compared to complementary DNA (cDNA) because they contain only the specific mutation and provide all the regulatory sequences required for in vivo gene expression. However, precisely mutated BACs are typically rare (∼1:1,000 to 1:100,000), making their isolation quite challenging. Although these BACs have been classically isolated by linking the mutation to additional genes, i.e., selectable markers, this approach is prone to false positives and is labor-intensive because it requires the subsequent removal of the selectable marker. We created Founder Principle-driven Enrichment (FPE), a method based on the population genetics "founder principle," to directly isolate rare mutant BACs, without any selectable marker, from liquid cultures via the polymerase chain reaction (PCR). Here, we provide a detailed description of FPE, including protocols for BAC recombineering and PCR screening. © 2017 by John Wiley & Sons, Inc.

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A One-Step Miniprep for the Isolation of Plasmid DNA and Lambda Phage Particles

Cited by 25 publications

References 33 publications

Live‐cell PCR and one‐step purification streamline DNA engineering

Live‐cell PCR and one‐step purification streamline DNA engineering

Efficient Disruption of Escherichia coli for Plasmid DNA Recovery in a Bead Mill

Direct Isolation of Seamless Mutant Bacterial Artificial Chromosomes

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