Plasmid DNA Purification

Introduction

Extraction of macromolecules such as DNA, RNA, and protein is one of the basic methods used in molecular biology. The process of extraction and purification of nucleic acids has evolved from being a complex, prolonged, and labor-intensive procedure. Nucleic acid purification technologies now give high sample outputs, increased yield and purity, and scalability of biomolecules with minimum cross-contamination. Over the recent years the automated systems designed for medium-to-large laboratories have also steadily grown in demand.

Preparation of Nucleic Acids

Preparation of nucleic acids begins with the process of sample collection (bacteria, animal tissue and cells, or plant tissue). Samples must be collected and handled properly to achieve high-quality nucleic acid regardless of the method used for DNA preparation. The path from sample collection to nucleic acid purification may involve sample collection, transport, archiving, storage, and purification of nucleic acids as a workflow (Figure 1).

Simple work flow for sample collection, transport, archiving, and DNA purification

Figure 1: Simple work flow for sample collection, transport, archiving, and DNA purification

Nucleic acid isolation: Overview

Isolation of DNA involves the lysis of cell membranes, removal of histone proteins, RNA, and lipids, and purification. Various methods available for extraction and purification are detailed in Table 1.

Table 1: Overview of methods for nucleic acid isolation

METHODS FEATURES
Conventional Method
Alkaline Extraction Method
(PLED35)
  • alkaline denaturation of high molecular weight chromosomal DNA
  • used to isolate plasmid DNA 
  • recovery process post centrifugation
Cesium Chloride (CsCl) Density Gradient Centrifugation
  • preparative density-gradient ultracentrifugation of DNA
  • used to isolate plasmid DNA
  • purified nucleic acid must be re-precipitated with alcohol
Oligp(dT)-Cellulose Chromatography
  • purification of RNA
  • large quantities of RNA from mammalian cells
Guanidinium Thiocyanate-Phenol-Chloroform Extraction
(30911)
  • a single step liquid – liquid extraction used for the extraction of RNA from cultured cells and most animal tissues
  • generates high yield in four hours
  • follow-up process done by precipitation with isopropanol
Solid-phase Nucleic Acid Extraction
Silica Matrices*
  • for selective DNA binding and DNA purification
  • purified DNA molecules can be eluted under low ionic strength (pH ≥7) later by using Tris-EDTA buffer or distilled water
Glass Particles
  • used to separate nucleic acid from other substances in the presence of chaotropic salts solution
Diatomaceous earth (kieselguhr or diatomite)
  • useful for the purification of plasmid and other DNA by immobilizing DNA onto particles in the presence of a chaotropic agent
  • DNA is eluted with a low salt buffer or in distilled water
Anion-Exchange Material
  • interaction between positively charged diethylaminoethyl cellulose (DEAE) groups on the resin’s surface and negatively charged phosphates of the DNA backbone

* GenElute products

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Plasmid DNA

Plasmids are significant tools in molecular biology for manipulating and decoding genetic information. They have evolved as key components in any cloning and biotechnological techniques as they are easier to manipulate.

Various methods have been developed for plasmid DNA purification. Sigma Life Science is committed to bringing you Greener Alternative Products, which adhere to one or more of The 12 Principles of Greener Chemistry. The GenElute products have Inherently Safer Chemistry, compared to the standard use of phenol and chloroform to perform DNA extractions.

GenElute™ Plasmid Miniprep Kit offered by Sigma-Aldrich is a simple, rapid, and cost-effective method for isolating plasmid DNA from E. coli cultures. The kit combines silica-based membrane technology and the convenience of a spin column format, and recovers up to 20 mg of high copy plasmid DNA per ml of overnight culture. The following are the main steps (Figure 2) in the isolation and purification of plasmid DNA using GenElute™ Plasmid Miniprep Kit.

  1. Bacterial cells are harvested via centrifugation, subjected to a modified alkaline-SDS lysis procedure, and the DNA adsorbed onto silica in the presence of high salts.
  2. Contaminants are then removed by a simple wash step.
  3. The bound DNA is eluted in water or Tris-EDTA buffer. The recovered plasmid DNA is predominately in its supercoiled form.
  4. The DNA is ready for use in applications such as restriction enzyme digestion, cloning, PCR, transformation, transcription, conventional and automated sequencing

Some of the plasmid DNA purification kits by Sigma Aldrich are tabulated in table 2. Detailed explanation of the different plasmid DNA purification methods can be found here:

/content/dam/sigma-aldrich/docs/Sigma/General_Information/2/plasmidpurification.pdf

Workflow for nucleic acid preparation and purification from plasmids

Figure 2: Workflow for nucleic acid preparation and purification from plasmids

Table 2:  Plasmid DNA purification kits by Sigma Aldrich

CATALOG PRODUCT NAME Features

PLN10

PLN70

PLN350

GenElute™ Plasmid Miniprep Kit
  • purify up to 20µg of plasmid DNA per ml of culture
  • purified plasmid DNA in less than 30 minutes for up to 24 preps
  • faster than gravity flow anion exchange columns
  • no detectable genomic DNA or RNA contamination
  • no phenol/chloroform extraction or alcohol precipitation required
  • contains additional wash buffer for use with enda+ E. coli bacterial strains (e.g., hb101 jm101, bl21)

NA0150S

NA0150

NA0160

NA0200S

NA0200

NA0300S

NA0300

NA0310

GenElute™ HP Plasmid Miniprep Kit
  • from harvested bacterial culture to pure plasmid DNA in 30 minutes or less
  • up to 25 µg (mini), 350 µg (midi), and 1.2 mg (maxi) yield of high-copy plasmid DNA
  • offers the flexibility of a vacuum or spin format
  • no phenol/chloroform extraction or alcohol precipitation required
  • kits are stable at room temperature for convenient storage
  • cost effective
PLED35 GenElute™ Endotoxin-free Plasmid Midiprep Kit
  • purify up to 250 mg endotoxin-free plasmid DNA (≤0.1 eu/μg DNA)
  • fast and simple, allowing up to 12 preps in less than 2 hours
  • faster than gravity flow ion exchange columns or magnetic bead preps
  • no expensive magnetic equipment required
NA0500 GenElute™ HP Plasmid Megaprep Kit
  • sophisticated – newest technology ensures improved performance
  • plentiful – yields 5 mgs of high-quality, endotoxin-free (≤0.1eu/μg) plasmid DNA in 90 minutes or less
  • convenient – vacuum format with no ethanol precipitation required

NA0400S

NA0400

NA0410

GenElute™ HP Endotoxin-free Plasmid Maxiprep Kit
  • fast – from harvested bacterial culture to pure plasmid DNA in 40 minutes
  • flexible – convenient vacuum format does not use gravity flow columns
  • high yields – up to 1.2 mg of high-copy plasmid DNA with ≤0.1eu/μg
NA0600 GenElute™ HP Endotoxin-free Plasmid Megaprep Kit
  • fast – from harvested bacterial culture to pure plasmid DNA in 90 minutes
  • convenient – vacuum format with no ethanol precipitation required
  • high yields – 5 mg of high-copy plasmid DNA (≤0.1eu/μg)
NA0800 GenElute™ HP Select Plasmid Gigaprep Kit
  • sophisticated – utilizes HP select technology, the premier plasmid purification method
  • high yields – 15 mg of high-quality, endotoxin-free (≤0.1eu/μg) plasmid DNA in 2 hours or less
  • convenient – vacuum format with no ethanol precipitation required
  • versatile – can be used to purify low-, medium-, and high-copy plasmid DNA
NA0100 PhasePrep™ BAC DNA Kit
  • typical DNA yields of 2-100μg from 5-500ml of overnight cultures
  • no phenol/chloroform extraction required
  • allows possible micro to maxi preps with the same kit

 

Table 3:  Related products by Sigma Aldrich

Catalog No. Product Name
93121-017 CloneStable®
90121-007 CloneStable®
NA1020 GenElute™ PCR Clean-Up Kit
PCR9601 GenElute™ 96 Well PCR Clean-Up Kit
PCR9604 GenElute™ 96 Well PCR Clean-Up Kit

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References

  1. Tan SC, Yiap BC. DNA, RNA, and protein extraction: the past and the present. J Biomed Biotechnol. 2009:574398.
  2. L. Buckingham and M. L. Flaws, Molecular Diagnostics: Fundamentals, Methods, & Clinical Applications, F.A. Davis, Philadelphia, Pa, USA, 2007.
  3. Weaver, RF. Molecular Biology, 2nd edition. McGraw Hill, San Francisco. p 93-94 (2002).

/content/dam/sigma-aldrich/docs/Sigma/General_Information/2/plasmidpurification.pdf  

https://www.csun.edu/~hcbio027/biotechnology/lec2/PL/pl.htm  

 

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