Plasmid DNA Purification using illustra™ plasmidPrep Mini Spin Kit

illustra™ plasmidPrep Mini Spin Kit utilizes a simple plasmid DNA purification protocol, employing a modified alkaline cell lysis procedure and a novel silica-based membrane. It is designed for the extraction and purification of general molecular biology grade plasmid DNA from E. coli. A modified alkaline lysis procedure is followed by clarification through centrifugation to remove precipitated genomic DNA, KDS, and other contaminants. No organic solvents are used; instead, chaotropic salts are included to denature protein components and promote the selective binding of plasmid DNA to the novel silica membrane (6, 7). Denatured contaminants are easily removed by subsequent centrifugation and washing steps. The purified plasmid DNA is eluted in a low-ionic-strength buffer, at a plasmid concentration suitable for most molecular biological applications. The steps for plasmid DNA purification using illustra™ plasmidPrep Mini Spin Kit are summarized below.

Materials

illustra™ mini columns, buffers, and collection tubes are provided with the kit

Absolute ethanol

Advance preparation

Warm lysis buffer type 8 to dissolve precipitate, if present. Store at room temperature.

Prior to first use, add absolute ethanol to the bottle containing wash buffer (check product instructions for volume). Mix by inversion, record completion on the label, and store upright and airtight.

Protocol

Note: Columns and buffers are NOT transferable between Cytiva kits in the illustra™ product range. Please ensure that you use the correct columns and buffers for your purification.

1. Harvest bacterial culture

Harvest bacteria by centrifugation and remove spent medium.

2. Lyse cells

Resuspend cells in an isotonic solution containing RNase. Add alkaline lysis buffer to denature genomic DNA and proteins. Neutralize the pH of the lysate with an acetate-buffered solution containing a chaotropic salt. Centrifuge the sample to pellet cellular debris, including genomic DNA, proteins, and lipids.

Cell resuspension can be achieved by vortexing, pipetting up/down, or by scraping the base of the microcentrifuge tube across the surface of an empty pipette tip box. Incomplete cell resuspension will result in reduced plasmid DNA recovery.

Vigorous mixing will shear genomic DNA, resulting in contamination. Do not allow the cell lysis reaction to exceed 5 min. Solution II contains NaOH, which will denature the plasmid DNA on prolonged incubation.

3. Bind plasmid DNA

Apply the cleared cellular lysate to an illustra™ plasmid mini column. Plasmid DNA binds to the silica membrane in the presence of chaotrope.

4. Wash (strain dependent)

This step is recommended for EndA⁺ E. coli strains such as HB101.

It removes residual nuclease activity and carbohydrates.

5. Wash and dry

Add buffer containing salt and ethanol to remove residual salts and other contaminants. Silica-bound plasmid DNA is dried to remove residual ethanol.

After centrifugation, if any of the wash buffer comes into contact with the bottom of the column, discard the flowthrough and recentrifuge for 30 s. The presence of contaminating ethanol in the eluted plasmid DNA may affect downstream applications. Therefore, care must be taken to ensure its complete removal.

6. Elute purified plasmid DNA

Elute plasmid DNA in a low-ionic-strength buffer containing EDTA.

Materials