Control of pH during plasmid preparation by alkaline lysis of Escherichia coli

Cloninger, Cheri; Felton, Marilyn; Paul, Bonnie; Hirakawa, Yasuko; Metzenberg, Stan

doi:10.1016/j.ab.2008.04.017

Cited by 11 publications

(8 citation statements)

References 9 publications

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“…1a). In the first step, to minimize eccDNA loss, we replaced the conventional unbuffered sodium hydroxide lysis, which may cause irreversible denaturation or breakage of DNA circles 11 , with a modified alkaline buffer at pH 11.8 to lyse the whole cells. In the second step, we used the rare cutter PacI restriction enzyme to linearize mtDNA before addition of an exonuclease (Plasmid-Safe ATP-dependent DNase) to digest linear DNA.…”

Section: An Efficient Eccdna Purification Methodsmentioning

confidence: 99%

See 1 more Smart Citation

eccDNAs are apoptotic products with high innate immunostimulatory activity

Wang

Djekidel

et al. 2021

Nature

142

204

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Extrachromosomal circular DNA elements (EccDNAs) have been described in the literature for several decades, and are known for their broad existence across different species 1 , 2 . However, their biogenesis, and functions are largely unknown. By developing a new circular DNA enrichment method, here we purified, and sequenced full-length eccDNAs with Nanopore sequencing. We found that eccDNAs are mapped across the entire genome in a close to random fashion, suggesting a biogenesis mechanism of random ligation of genomic DNA fragments. Consistently, we found that apoptosis inducers can increase eccDNA generation, which is dependent on apoptotic DNA fragmentation followed by ligation by the DNA ligase 3. Importantly, we demonstrated that eccDNAs can function as potent innate immunostimulants in a sequence-independent, but circularity, and cytosolic DNA sensing Sting-dependent fashion. Collectively, our study not only revealed the origin, biogenesis, and immunostimulant function of eccDNAs, but also uncovered their sensing pathway and potential clinical implications in immune response.

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Section: An Efficient Eccdna Purification Methodsmentioning

confidence: 99%

“…1a ). In the first step, to minimize eccDNA loss, we changed the unbuffered sodium hydroxide that can cause denaturation or breakage of DNA circles 11 to modified alkaline lysis at pH 11.8. In the second step, we used a rare-cutter Pac I restriction enzyme to linearize mtDNA before an exonuclease (P.S.…”

Section: An Efficient Eccdna Purification Methodsmentioning

confidence: 99%

eccDNAs are apoptotic products with high innate immunostimulatory activity

Wang

Djekidel

et al. 2021

Nature

142

204

View full text Add to dashboard Cite

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“…Cultures producing PCR products of appropriate size were transferred and grown in fresh LB broth with ampicillin (100 μg mL −1 ) for 16 h at 37°C and 250 rpm. Plasmids were then extracted via alkaline lysis (Cloninger et al, 2008) and bidirectionally sequenced on an ABI 3730 using M13 forward and reverse primers.…”

Section: Methodsmentioning

confidence: 99%

Nitrification and Denitrification Gene Abundances in Swine Wastewater Anaerobic Lagoons

Ducey¹,

Shriner²,

Hunt³

2011

J of Env Quality

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Although anaerobic lagoons are used globally for livestock waste treatment, their detailed microbial cycling ofN is only beginning to become understood. Within this cycling, nitrification can be performed by organisms that produce the enzyme ammonia monooxygenase. For denitrification, the reduction of nitrite to nitric oxide can be catalyzed by two forms of nitrite reductases, and N,O can be reduced by nitrous oxide reductase encoded by the gene nosZ The objectives of this investigation were to (i) quantify the abundance of the amoA, nirK, nirS, and nosZ genes; (ii) evaluate the influence of environmental conditions on their abundances; and (iii) evaluate their abundance relative to denitrification enzyme activity (DEA). Samples were analyzed via real-time quantitative polymerase chain reaction and collected from eight typical, commercial anaerobic, swine wastewater lagoons located in the Carolinas. The four genes assayed in this study were present in all eight lagoons. Their abundances relative to total bacterial populations were 0.04% (amoA), 1.33% (nirS), 5.29% (nirK), and 0.27% (nosZ). When compared with lagoon chemical characteristics, amoA and nirK correlated with several measured variables. Neither nirS nor nosZ correlated with any measured environmental variables. Although no gene measured in this study correlated with actual or potential DEA, nosZ copy numbers did correlate with the disparity between actual and potential DEA. Phylogenetic analysis ofnosZdid not reveal any correlations to DEA rates. As with other investigations, analyses of these genes provide useful insight while revealing the underlying greater complexity of N cycling within swine waste lagoons.

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“…Therefore, in this work, we develop a bacterial single-cell lysis protocol as a guideline for MDA-based bacterial SC-WGA in microfluidic platforms that produces >25 ng of genomic DNA per cell, sufficient for downstream library preparation. This on-chip protocol combines three primary bacterial lysis methods which include thermal [ 15 , 43 , 44 ], enzymatic [ 45 , 46 ] and chemical lysis [ 47 , 48 , 49 ] and was tested on both gram-positive and gram-negative bacterial species for subsequent on-chip SC-WGA. In this study, Corynebacterium glutamicum was used as a typical gram-positive model whose cell wall is thicker than most gram-negative species.…”

Section: Introductionmentioning

confidence: 99%

The Development of an Effective Bacterial Single-Cell Lysis Method Suitable for Whole Genome Amplification in Microfluidic Platforms

et al. 2018

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Single-cell sequencing is a powerful technology that provides the capability of analyzing a single cell within a population. This technology is mostly coupled with microfluidic systems for controlled cell manipulation and precise fluid handling to shed light on the genomes of a wide range of cells. So far, single-cell sequencing has been focused mostly on human cells due to the ease of lysing the cells for genome amplification. The major challenges that bacterial species pose to genome amplification from single cells include the rigid bacterial cell walls and the need for an effective lysis protocol compatible with microfluidic platforms. In this work, we present a lysis protocol that can be used to extract genomic DNA from both gram-positive and gram-negative species without interfering with the amplification chemistry. Corynebacterium glutamicum was chosen as a typical gram-positive model and Nostoc sp. as a gram-negative model due to major challenges reported in previous studies. Our protocol is based on thermal and chemical lysis. We consider 80% of single-cell replicates that lead to >5 ng DNA after amplification as successful attempts. The protocol was directly applied to Gloeocapsa sp. and the single cells of the eukaryotic Sphaerocystis sp. and achieved a 100% success rate.

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Control of pH during plasmid preparation by alkaline lysis of Escherichia coli

Cited by 11 publications

References 9 publications

eccDNAs are apoptotic products with high innate immunostimulatory activity

eccDNAs are apoptotic products with high innate immunostimulatory activity

Nitrification and Denitrification Gene Abundances in Swine Wastewater Anaerobic Lagoons

The Development of an Effective Bacterial Single-Cell Lysis Method Suitable for Whole Genome Amplification in Microfluidic Platforms

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