Plasmid DNA

Purification of plasmid DNA

The need for efficient plasmid DNA purification methods has grown significantly during the past decade. In addition to basic research plasmid DNA is used

• in gene therapy
• for vaccine development
• for gene expression studies
• or for gen-knock-in or knock-out studies (i.e CRISPR/Cas) for gene expression

In the following paragraphs we describe crutial steps for the purification of plasmid DNA. If you have further questions, please contact our MN TechSupport Team.
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NucleoBond Xtra plasmid kits - fast, clean and reliable

• Proven anion-exchange technology for highest yields and quality
• Optimized column design for minimum risk of clogging
• NucleoBond Xtra Maxi, yields of up to 1000 μg of ultrapure plasmid DNA
• Accelerated procedure with NucleoSpin Finalizer for a complete prep in approximately 30 min.

Crucial steps

Yield and quality of the plasmid DNA are highly dependent on the bacterial culture used as input. Cultivation strategies (e. g., culture medium, culture conditions, culture duration, cell strain and many further factors) as well as plasmid design (e. g., copy number, insert size, insert sequence, selection factors, dimer formation and many further factors) determine the plasmid quality primarily. The preparation itself has no mayor influence on the general plasmid characteristics (e. g., copy number per cell or ratio of supercoiled plasmid to nicked plasmid). Nevertheless there are crucial steps which might influence the outcome concerning plasmid DNA yield and contaminations.
Especially the lysis procedure has got a significiant influence on the resulting yield and the presence of co-purified contaminants (e. g., endotoxins, host RNA, host genomic DNA, host proteins).
The crucial steps and corresponding considerations are:

Determination of cell input

It is crucial to use the correct amount of cells as input. Using too many cells will result in

• insufficient lysis
• reduced plasmid DNA yield
• increased presence of contaminants like host protein, host genomic DNA and host RNA.

Estimating the amount of cellular input

To estimate the amount of cells it is recommended to either measure the optical density of the bacterial culture at 600 nm (OD600) before pelleting the cells or to use the wet weight of the bacterial cell pellet after centrifugation.

The solution

Effect of excess cell input on plasmid yield

Excess cell input decreases the plasmid DNA yield /µg drastically as shown in the gel picture. A misbalance of the pelleted culture volume and the OD is shown in the decreasing yields of plasmid DNA /µg. The graph on the right shows the difference between the theroretical plasmid yield (orange) and the acutal purified plasmid yield (blue).

Adaptation for excess amount of cells

By rule of thumb, calculate the necessary lysis buffer volumes for RES, LYS and NEU as follows:

Contact the MN Tech Support Team for further information and for tips and tricks to modify the precedure to process larger cell pellets than recommended.
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Resuspension

Resuspend the cells in the given or calculated volume of resuspension buffer. Take care that the cells of the pellet are singled out as completely as possible. The cell pellet might dislodge from the reaction tube wall and thus become difficult to see. If in doubt, use a pipette to pipette up and down. Residual cell clumps will clog the pipette tip and can be felt by an increased pipetting resistance.

Impact on plasmid yield

The graph and agarose gel picture show the effect of incompletely resuspended cells (corresponding samples in the pictures above).

Lysis - important factors

Precipitation of buffers

A common root cause for low plasmid yield is precipitated sodium dodecyl sulfate (SDS), a component of the lysis buffer. SDS might be precipitated due to storage and transport at low ambient temperatures or in refrigerated warehouses.

The solution

Incubate the bottle for several minutes at 30–40 °C and mix well until SDS is redissolved.

Effects of precipitated lysis buffer on plasmid yield

Sodium dodecyl sulfate (SDS) leads to the solubilization of cell wall components such as proteins or phospholipids, leading to the release of plasmid DNA. If SDS is precipitated, this process is unsuccessfully, plasmid DNA is not released from the bacteria, and the plasmid yield is tremendously affected. The graph and agarose gel picture show the result of SDS precipitation in Buffer LYS. Plasmid DNA is hardly detectable on the agarose gel and via spectrophotometry

Shearing forces

Furthermore, invert the samples gently during the lysis step. Shearing forces induced by harsh treatment will result in a fragmentation of host genomic DNA. Fragmented DNA is less efficiently removed during the neutralization step and will be co-purified with the plasmid DNA

Neutralization

During the neutralization step, host genomic DNA and most host proteins are removed and binding conditions are established. Invert the samples gently. If the lysis buffer contained a LyseControl (blue color), continue until no spots of blue color are visible anymore.