High Pure Plasmid Isolation Kit

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 A ₆₀₀ 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.

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

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)

For life science research only. Not for use in diagnostic procedures.

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.