Plasmid DNA Purification

plasmid 50 min plasmid dna extraction

E.Z.N.A. Plasmid DNA Mini Kit I | Plasmid DNA Purification

The E.Z.N.A.® Plasmid DNA Mini Kit I is designed to isolate up to 25 µg of high-quality plasmid DNA from 1-5 mL bacterial cultures in less than 30 minutes. Plasmid DNA purification follows the alkaline-lysis method and is simplified with HiBind® Mini Column technology into three quick steps: Bind, Wash, and Elute. Purified plasmid DNA is immediately ready for a wide variety of downstream applications such as routine screening, restriction enzyme digestion, transformation, PCR and DNA sequencing.

The E.Z.N.A.® Plasmid DNA Mini Kit is available in two formats – Q-spin (D6942) and V-spin (D6943). D6942 (Q-spin) features columns that are capless whereas D6943 (V-spin) includes columns that have a cap attached. The columns are otherwise identical in use and application and can be used in both vacuum or centrifugation protocols.

  • Rapid – Purification of plasmid DNA in less than 30 minutes
  • Safe – No Phenol/chloroform extractions
  • Versatile – Spin and vacuum formats available
  • High-quality – DNA is suitable for a variety of downstream applications
plasmid 50 min plasmid dna extraction
      plasmid 50 min plasmid dna extraction

 

Product Data

Plasmid DNA Yield and Purity from 4 mL Bacterial Culture using E.Z.N.A.® Plasmid DNA Mini Kit I and a competing product from company Q

Figure 1.   pGEM plasmid was purified from 4 mL DH5α cultures harboring the plasmid and eluted in 50 µL volume using kits from Omega Bio-tek and Company Q according to manufacturer’s recommended protocols. Plasmid DNA concentration was determined by optical density measurements using Thermo Scientific’s NanoDrop™ 2000c system.

The direct extraction of plasmid DNA containing antibiotic resistance genes from complex samples is imperative when studying plasmid-mediated antibiotic resistance from a One Health perspective, in order to obtain a wide representation of all the resistance plasmids present in these microbial communities. There are also relatively few bacterial species from natural environments which can be cultured in vitro. Extracting plasmids from the cultivable fraction of these complex microbiomes may only represent a fraction of the total antibiotic resistance plasmids present. We compared different methods of plasmid extraction from broiler cecal samples, whose resistance could be expressed in a human pathogen—Escherichia coli. We found that kits designed for DNA extraction from complex samples such as soil or feces did not extract intact plasmid DNA. Commercial kits specific for plasmid extraction were also generally unsuccessful, most likely due to the complexity of our sample and intended use of the kits with bacterial culture. An alkaline lysis method specific for plasmid extraction was ineffective, even with further optimization. Transposon-aided capture of plasmids (TRACA) allowed for the acquirement of a small range of resistance plasmids. Multiple displacement amplification provided the broadest range of resistance plasmids by amplifying all extracted circular plasmid DNA, but the results were not reproducible across all samples. Exogenous plasmid isolation enabled the extraction of resistance plasmids from the microbial fraction by relying on the mobility of the plasmids in the sample. This was the most consistent method from which we obtained a range of resistance plasmids from our samples. We therefore recommend the use of the exogenous plasmid isolation method in order to reliably obtain the greatest representation of the total antibiotic resistance plasmidome in complex samples. While this method has limitations, it is one which will vastly increase our current knowledge of antibiotic resistance plasmids present in complex environments and which are capable of transferring to a human and animal pathogen and environmental contaminant.

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