Simple, inexpensive and RNase-free purification of plasmid DNA by fractional precipitation with isopropanol

BioTechniques ◽

10.2144/btn-2021-0018 ◽

2021 ◽

Author(s):

Viiu Paalme ◽

Mart Speek

Keyword(s):

Quality Control ◽

Column Chromatography ◽

Plasmid Dna ◽

Fractional Precipitation ◽

Alkaline Lysis ◽

Bacterial Extract ◽

Total Rna ◽

Spin Column ◽

Commercial Kits

We present a modified alkaline lysis method for purification of plasmid DNA (pDNA) from bacterial extract using fractional precipitation with isopropanol (FPI). This method includes two successive precipitations with 0.33 and 0.36 volumes of isopropanol and separates pDNA from total RNA and most of the lipopolysaccharides. Using different quality control tests, we demonstrate that plasmids purified with FPI show superior quality compared to plasmids prepared with commercial kits based on spin-column chromatography.

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Filter paper-based spin column for low throughput nucleic acid purification

10.1101/392696 ◽

2018 ◽

Author(s):

Rui Shi ◽

Ramsey S. Lewis ◽

Dilip R. Panthee

Keyword(s):

Nucleic Acid ◽

Nucleic Acids ◽

Filter Paper ◽

Genomic Dna ◽

Total Rna ◽

Binding Material ◽

Pcr Product ◽

Spin Column ◽

Nucleic Acid Purification ◽

Commercial Kits

AbstractWe describe a method of recharging used spin column or assembling homemade spin column using filter paper as binding material for low throughput nucleic acid purification. We evaluated the efficiency of filter paper based spin columns in the purification of different type of nucleic acids. For instance, by following protocols of respective commercial kits, we found that filter paper to be a useful binding material for purification of many types of nucleic acids, including plant genomic DNA, plant total RNA, PCR product, and DNA from agarose gels. We also found that filter paper has a weak binding affinity to plasmid DNA in tested miniprep protocols. Also, we present the protocols of using filter paper recharged spin column or homemade spin column for low throughput purification of plant genomic DNA and plant total RNA with commercial kit buffer leftover and less expensive homemade buffer.

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The Macerprep: a minimalist kit- and enzyme-free high-yield miniprep utilising alkaline lysis and alkaline hydrolysis principles

10.1101/2020.08.13.249607 ◽

2020 ◽

Author(s):

Ian Hu

Keyword(s):

Molecular Biology ◽

Alkaline Hydrolysis ◽

Plasmid Dna ◽

Dna Isolation ◽

Operation Time ◽

High Yield ◽

Alkaline Lysis ◽

Commercial Kits ◽

Dna Isolation Protocol ◽

Over Time

ABSTRACTThe commercialisation of miniprep kits supplanted the original alkaline lysis method for plasmid DNA preparation, and had remained relatively unchanged for almost two decades. The Miraprep substantially improved the yields of miniprep kits. However, the method still relies on commercial kits, which can be a burden financially to certain projects. Additionally, Pronobis et al. also identified loss of RNAse activities in miniprep kits over time. The present novel plasmid DNA isolation protocol addresses the two issues mentioned above utilising alkaline lysis and alkaline hydrolysis principles. With a largely identical workflow and operation time, the Macerprep will significantly reduce costs of establishing new laboratories as well as maintenance of running molecular biology laboratories.

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Plasmid DNA Extraction from E. coli Using Alkaline Lysis Method

BIO-PROTOCOL ◽

10.21769/bioprotoc/bio101.30 ◽

2011 ◽

Vol 1 (3) ◽

Cited By ~ 1

Author(s):

Fanglian He

Keyword(s):

Dna Extraction ◽

Plasmid Dna ◽

Alkaline Lysis ◽

E Coli

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Multiple Displacement Amplification as a Solution for Low Copy Number Plasmid Sequencing

Frontiers in Microbiology ◽

10.3389/fmicb.2021.617487 ◽

2021 ◽

Vol 12 ◽

Author(s):

Kuan Yao ◽

Narjol González-Escalona ◽

Maria Hoffmann

Keyword(s):

Antibiotic Resistance ◽

Single Molecule ◽

Plasmid Dna ◽

Copy Number ◽

Sequence Data ◽

Multiple Displacement Amplification ◽

Fast Method ◽

Alkaline Lysis ◽

Low Copy Number ◽

Long Read

Plasmids play a major role in bacterial adaptation to environmental stress and often contribute to antibiotic resistance and disease virulence. Although the complete sequence of each plasmid is essential for studying plasmid biology, most antibiotic resistance and virulence plasmids in Salmonella are present only in a low copy number, making extraction and sequencing difficult. Long read sequencing technologies require higher concentrations of DNA to provide optimal results. To resolve this problem, we assessed the sufficiency of multiple displacement amplification (MDA) for replicating Salmonella plasmid DNA to a satisfactory concentration for accurate sequencing and multiplexing. Nine Salmonella enterica isolates, representing nine different serovars carrying plasmids for which sequence data are already available at NCBI, were cultured and their plasmids isolated using an alkaline lysis extraction protocol. We then used the Phi29 polymerase to perform MDA, thereby obtaining enough plasmid DNA for long read sequencing. These amplified plasmids were multiplexed and sequenced on one single molecule, real-time (SMRT) cell with the Pacific Biosciences (Pacbio) Sequel sequencer. We were able to close all Salmonella plasmids (sizes ranged from 38 to 166 Kb) with sequencing coverage from 24 to 2,582X. This protocol, consisting of plasmid isolation, MDA, and multiplex sequencing, is an effective and fast method for closing high-molecular weight and low-copy-number plasmids. This high throughput protocol reduces the time and cost of plasmid closure.

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