Plasmid DNA Purification

Introduction to plasmid purification

Extraction of macromolecules such as DNA, RNA, and protein is common in molecular biology research. The process of extraction and purification of nucleic acids has evolved from being a complex and labor-intensive procedure. Current nucleic acid purification methods provide high sample outputs, purity, and scalability of biomolecules with minimum cross-contamination. Automated systems designed for medium-to-large laboratories are also commonly used.

Preparing nucleic acids for plasmid purification

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).

Figure 1. Workflow for sample collection, transport, archiving, and DNA purification.

Nucleic acid isolation

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

Plasmid DNA

Plasmids are small, circular, double-stranded DNA used 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. We are 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.

Our GenElute™ Plasmid Miniprep Kit 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 our plasmid DNA purification kits  are tabulated in Table 2. Detailed explanation of the different plasmid DNA purification methods (PDF).

Figure 2. Workflow for nucleic acid preparation and purification from plasmids. PCR DNA clean-up: product numbers NA1020 and PCR9604

Table 2.Plasmid DNA purification kits

Product No.	Product Name	Features
PLN10 PLN70 PLN350	GenElute™ Plasmid Miniprep Kit	purify up to 20 µg 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 of 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 mg 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 ≤0.1 eu/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 (≤0.1 eu/μg)
NA0800	GenElute™ HP Select Plasmid Gigaprep Kit	sophisticated – uses HP select technology, the premier plasmid purification method high yields – 15 mg of high-quality, endotoxin-free (≤0.1 eu/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 2-100 μg from 5-500 mL of overnight cultures no phenol/chloroform extraction required allows possible micro to maxi preps with the same kit

Table 3.PCR clean-up products

Product No.	Product Name
NA1020	GenElute™ PCR Clean-Up Kit
PCR9604	GenElute™ 96 Well PCR Clean-Up Kit

References

1. Tan SC, Yiap BC. 2009. DNA, RNA, and Protein Extraction: The Past and The Present. Journal of Biomedicine and Biotechnology. 20091-10. https://doi.org/10.1155/2009/574398

2. Buckingham L, Flaws ML. 2007. Flaws, Molecular Diagnostics: Fundamentals, Methods, & Clinical Applications. Philadelphia, USA: F.A. Davis Company.

3. Weaver R. 2002. Molecular Biology . 2nd edition. San Francisco: McGraw Hill.