Ion Exchange Chromatography
Ion exchange chromatography is employed by the Genopure Plasmid Midi Kit and the Genopure Plasmid Maxi Kit. The isolation method is based on a modified alkaline lysis protocol and can be divided into the following steps also outlined in Figure 1:
- Harvest and disruption of the bacterial cells
- Precipitation of the bacterial "chromosomal" DNA
- Clarification of the bacterial lysate
- Removal of residual impurities by wash steps
- Elution of the plasmid DNA by high salt conditions
- Concentration and salt removal by alcohol precipitation
The isolation method is optimized for cultures grown in LB media; other rich media may require increased volumes of Suspension-, Lysis-, and Neutralization Buffer, and an additional wash step. The isolation procedure is suitable for all plasmid sizes; lysates of larger constructs (up to 100 kb) should be cleared by filtration to avoid shearing.
The yield of plasmid DNA preparations is dependent on several parameters, e.g., quality of the bacterial culture growth, amount of used culture suspension for the preparation, plasmid type used etc. As a rule of thumb the typical yield of a high copy number plasmid is about 3 - 5 µg of DNA per ml of original bacterial culture (pUC, pTZ, pGEM in common host strains like XL-1 blue, HB101, JM 109). The typical yield of low copy number plasmids is about 0.2 - 1 µg of DNA per ml of original bacterial culture.
The Genopure kits are supplied with folded filters to eliminate the time-consuming centrifugation step after the alkaline lysis. In approximately 2 minutes (midi) or 10 minutes (maxi), respectively, of unattended running cellular debris and potassium dodecylsulphate precipitates are held back by the filter thereby avoiding shearing of large DNA constructs. Besides the significant reduction of preparation time another advantage of filtration is that even small SDS precipitates which cannot be separated by conventional centrifugation are completely removed.