Description
General description
Low to medium throughput, mini scale, plasmid isolation.
The High Pure Plasmid Isolation Kit isolates purified plasmid DNA in small quantities using the alkaline lysis method, a commonly used method that generates highly purified plasmid DNA from E. coli, free of RNA contamination.
Nucleic acids bind to the surface of the glass fiber fleece in the presence of a chaotropic salt (guanidine HCl). This allows the High Pure filter tube to specifically immobilize nucleic acids (both DNA and RNA) while they are freed of contaminants.
Capacity: The High Pure Spin Filter Tubes hold up to 700 μL sample volume.
Sample: 0.5 - 4.0 mL cultures of E. coli (harvested at a density of 1.5 - 5.0 A ₆₀₀ units/mL)
Application
The High Pure Plasmid Isolation Kit prepares up to 15 μg purified plasmid DNA from bacterial cultures, that can be used directly in most molecular biology applications:
PCR
Sequencing
Cloning
In vitro transcription
Restriction enzyme digests
Random primed labeling
Features and Benefits
Quickly purify up to 24 plasmid samples in Minimize DNA loss with a kit that removes contaminants without precipitation or other handling steps that degrade DNA.
Improve reliability and reproducibility in downstream applications with a kit that removes RNA and other impurities that cause plasmid DNA to behave unpredictably.
Eliminate the use of hazardous organic compounds such as cesium chloride, phenol, chloroform, and ethidium bromide.
Preparation Note
The kit relies on alkaline lysis to release plasmid DNA from bacteria. RNase removes all RNA in the lysate. After cellular debris and (entrapped) genomic DNA are removed by centrifugation, the remaining supernatant is mixed with a chaotropic salt and applied to the glass fiber fleece in a High Pure Spin Filter Tube. Under the buffer conditions used in the procedure, the plasmid binds to the glass fiber fleece, while contaminating substances (salts, proteins, and other cellular contaminants) do not. Brief wash-and-spin steps readily remove these contaminants. Once purified, the plasmid can be easily eluted in a small volume of low-salt buffer or water.
Analysis Note
Plasmid pUC19 (4.5 μg) is purified from a 1.5 mL suspension of E. coli JM₈₃, which was grown in LB-medium with ampicillin for 16 hours, to a cell density of 5 A₆₀₀ units/mL. 1 μg of the purified plasmid DNA is incubated for 1 hour at +37°C with 5 units of the restriction endonuclease Eco RI and then analyzed by agarose gel electrophoresis. The isolated plasmid DNA is as sensitive to restriction endonuclease digestion as plasmid DNA isolated by CsCl density centrifugation.
Other Notes
High Pure Technology and Silica Adsorption Kits
Suspension Buffer
RNase A, dry powder
Lysis Buffer
Binding Buffer
Wash Buffer I
Wash Buffer II
Elution Buffer
High Pure Spin Filter Tubes (containing glass fiber fleece)
Collection Tubes
For life science research only. Not for use in diagnostic procedures.
