Working with Plasmids
Lysis of bacterial cells for plasmid purification
Alkaline lysis technique for plasmid DNA
Alkaline lysis, a crucial method in molecular biology for the efficient extraction of plasmid DNA from chromosomal DNA, was pioneered by Birnboim and Doly. It utilizes a simple, four-step procedure that takes advantage of the supercoiled characteristic of plasmid DNA to purify it from bacterial cells. Known for its straightforwardness and efficacy in DNA extraction, this technique is highly valued for its simplicity and efficiency.
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Resuspend harvested bacterial cells in Tris·Cl–EDTA buffer containing RNase A.
Tip: Ensure that bacteria are resuspended completely, leaving no cell clumps to maximize the number of cells exposed to the lysis reagents.
Tip: For large-scale purification of low-copy plasmids, for which larger culture 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 the solution appears very viscous after adding lysis buffer (NaOH/SDS) and is still 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 now allow the lysis to proceed for longer than 5 minutes. This is optimal for release of 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.
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Clear the lysate by either centrifugation or filtration to precipitate the debris.
Checking the buffer is sufficiently mixed – LyseBlue Reagent
LyseBlue is a color indicator that makes it easier to see if the buffer is optimally mixed. This prevents common handling errors that lead to inefficient cell lysis and incomplete precipitation of SDS, genomic DNA and cell debris. This makes LyseBlue ideal for researchers starting out with plasmid DNA preparations and experienced scientists who want to ensure maximum product yield. LyseBlue reagent is optional and not required to perform plasmid DNA preparations successfully.
LyseBlue can be added to the resuspension buffer (Buffer P1) bottle before use, or you can add smaller amounts of LyseBlue to aliquots of Buffer P1 for visual control of single plasmid DNA preparations.LyseBlue reagent should be added to Buffer P1 at a ratio of 1:1000 to achieve the required working concentration (e.g., 10 μL LyseBlue into 10 mL Buffer P1). LyseBlue precipitates after addition into Buffer P1. This precipitate will completely dissolve after addition of Buffer P2. Make sure you shake Buffer P1 before you use it to resuspend the LyseBlue particles, then carry out the plasmid DNA preparation as usual.
Note: You must make sufficient LyseBlue/Buffer P1 working solution for the number of plasmid DNA preps you want to perform.
After adding Buffer P2 to Buffer P1, the color of the suspension will change to blue. Mixing should result in a homogeneously colored suspension. If the suspension contains localized regions of colorless solution or brownish cell clumps are still visible, continue mixing the solution until you get a homogeneously colored suspension.
Upon addition of neutralization buffer (Buffer N3), LyseBlue turns colorless. The presence of a homogeneous solution with no traces of blue indicates that SDS from the lysis buffer has been effectively precipitated.
Other lysis methods
A number of other methods have been described for lysis E. coli cells (1, 6). Some of these methods were developed for other applications and may not be suitable for plasmid DNA preparation.
- Lysis with detergent: Bacterial cells are lysed by treatment with an ionic detergent (e.g., SDS)
- Mechanical lysis: Bacterial cells are lysed by mechanical disruption (e.g., by sonification).
- Boiling lysis and enzymatic digestion: Bacterial cells are treated with lysozyme to weaken the cell walls and then lysed by heating in a boiling water bath for ~1 minute.
Lysis of bacteria other than E. coli
Clearing of bacterial lysates using QIAfilter Cartridges
QIAfilter Cartridges are special filtration units designed to replace the centrifugation step after alkaline lysis of bacterial cells.
After lysis and neutralization, the lysate is transferred directly in the QIAfilter Cartridge. After incubation, pass the liquid through the filter to clear the lysate. This only takes a matter of seconds and will completely remove any insoluble complexes containing chromosomal DNA, salt, detergent and proteins which may have formed during the neutralization step.
QIAfilter Cartridges allow you to clear bacterial lysates more efficiently than conventional centrifugation. In addition, small-sized SDS precipitates that cannot be separated by centrifugation are completely removed using QIAfilter cartridges.
DNA binding and washing on the QIAGEN-tip
Once you have your cleared lysate, load it onto a pre-equilibrated QIAGEN-tip gravity flow. The salt and pH conditions of the lysate and the superior selectivity of the QIAGEN resin ensure that only plasmid DNA binds, while degraded RNA, cellular proteins and metabolites will not be retained and appear in the flow-through fraction.
Wash the QIAGEN-tip with medium-salt buffer (Buffer QC) to completely remove any remaining contaminants, such as traces of RNA and protein (e.g., RNase A), without affecting the binding of plasmid DNA. Buffer QC also disrupts nonspecific interactions and allows removal of nucleic acid-binding proteins without using phenol. The low concentration of alcohol in the wash buffer eliminates nonspecific hydrophobic interactions, further enhancing the purity of the bound DNA/. Efficiently elute the plasmid DNA from the QIAGEN-tip with high-salt buffer (Buffer QF or QN)Desalting and concentration by centrifugation
Desalt and concentrate your eluted plasmid DNA by isopropanol precipitation. You must carry out the precipitation at room temperature to minimize coprecipitation of salt.
After centrifuging, wash the DNA pellet in 70% ethanol to remove residual salt and replace the isopropanol with ethanol, which is more volatile and easily removed. After this, briefly air-dry the purified DNA and redissolve in a small volume of TE buffer, pH 8.0 or Tris·Cl, pH 8.5.
It is then ready for transfection, gene editing, sequencing, labeling, cloning or any other experimental procedure.
Desalting and concentration by QIAprecipitator module (only available in HiSpeed Plasmid Kits)
Once you’ve mixed your eluted plasmid DNA with isopropanol, it could be applied to the QIAprecipitator module using the syringe provided in the kit. The module traps precipitated DNA while the isopropanol mixture flows through.
Tip: We also recommend that you carry out an additional ethanol wash step to maximize DNA purity.
The precipitated DNA will be trapped in the QIAprecipitator as a thin layer. Thoroughly dry the module and remove any ethanol by simply pushing air through the QIAprecipitator with a syringe. Elute the DNA from the QIAprecipitator into a microcentrifuge tube with Buffer TE provided in the kit. Alternatively, you can use any common buffer or water.
You should store the DNA at –20°C when eluted with water, as the DNA may degrade without a buffering and chelating agent. The DNA is then ready to use directly for transfection, sequencing, labeling, cloning or any other experimental procedure.
Learn how to troubleshoot the different steps of the plasmid purification procedure by agarose gel electrophoresis.
Simple, ultrafast, large-scale purification
To perform ultrafast, large-scale purification of up to 10 mg of highly pure plasmid DNA, try the QIAGEN Plasmid Plus Kits.
Using the vacuum manifold, you can purify up to 24 samples in parallel, reducing your hands-on time. Even with low elution volumes, you still get high yields of concentrated plasmid DNA for direct use without ethanol precipitation. The QIAGEN Plasmid Plus Kits also include a dedicated wash buffer to reduce endotoxin contamination.
The plasmid DNA you get using this kit is highly suitable for many applications, including transfection into sensitive cell lines.
