Working with Plasmids
Lysis of bacterial cells for plasmid purificationEffective lysis of bacterial cells is a key step in plasmid isolation as DNA yield and quality depend on the quality of cell lysate used for the purification.
Alkaline lysis is one of the most commonly used methods for lysing bacterial cells prior to plasmid purification (4, 5). Production of alkaline lysates involves four basic steps (see figure The principle of alkaline lysis).
- Resuspend harvested bacterial cells in Tris·Cl–EDTA buffer containing RNase A.
Tip: Ensure that bacteria are resuspended completely leaving no cell clumps in order to maximize the number of cells exposed to the lysis reagents.
Tip: For large-scale purification of low-copy plasmids, for which larger cultures volumes are used, it may be beneficial to increase the lysis buffer volumes in order to increase the efficiency of alkaline lysis and thereby the DNA yield.
- Lyse cells using NaOH/SDS. Sodium dodecyl sulfate (SDS) solubilizes the phospholipid and protein components of the cell membrane, leading to lysis and release of the cell contents. NaOH denatures the chromosomal and plasmid DNA, as well as proteins. The presence of RNase A ensures that liberated cellular RNA is digested during lysis.
Tip: If after addition of lysis buffer (NaOH/SDS) the solution appears very viscous and is difficult to mix, this indicates excess biomass in the lysate step. This results in insufficient cell lysis and it is recommended to double the amount of lysis and neutralization buffers used.
Tip: Avoid vigorous stirring or vortexing of the lysate as this can shear the bacterial chromosome, which will then copurify with the plasmid DNA. The solution should be mixed gently but thoroughly by inverting the lysis vessel 4–6 times.
Tip: Do not allow the lysis to proceed for longer than 5 minutes. This is optimal for release of the plasmid DNA, while avoiding irreversible plasmid denaturation.
- Neutralize the lysate by adding acidic potassium acetate. Note: The high salt concentration causes potassium dodecyl sulfate (KDS) to precipitate, and denatured proteins, chromosomal DNA, and cellular debris are coprecipitated in insoluble salt-detergent complexes. Plasmid DNA, being circular and covalently closed, renatures correctly and remains in solution.
Tip: Precipitation can be enhanced by using chilled neutralization buffer and incubating on ice.
- Clear the lysate by either centrifugation or filtration, to precipitate the debris.
Note: Purification of plasmid DNA from cleared bacterial lysates was traditionally performed using cesium chloride (CsCl) ultracentrifugation. Today, a variety of commercially available plasmid purification kits offer easy procedures for different throughput requirements and applications.
Other lysis methods
A number of other methods have been described for lysing bacterial cells (1, 6). Some of these methods were developed for other applications and may not be suitable for plasmid DNA preparation.
- Boiling lysis: Bacterial cells are treated with lysosome to weaken the cell walls and then lysed by heating in a boiling water bath for ~1 minute.
- Lysis with detergent: Bacterial cells are lysed by treatment with and ionic detergent (e.g., SDS) or a nonionic detergent (e.g., Triton X-100).
- Mechanical lysis: Bacterial cells are lysed by mechanical disruption (e.g., by sonification).
- Enzymatic digestion: Some lysis methods include treatment of bacteria with enzymes such as lysozyme which assist in weakening cell walls.
Lysis of bacteria other than E. coli
Isolation of plasmid DNA from bacteria other than E. coli usually requires modifications to the lysis procedure in order to optimize lysis conditions for the particular species.