General Considerations for Plasmid DNA Preparation

The yield and purity of isolated plasmid DNA will be influenced not only by the method of isolation, that is, whether a commercially available purification kit is used, but also by a number of external factors, for example, culture cell density, duration of growth, medium used, the type of plasmid (high or low copy number), the size of the insert, and the host strain used.

Factors affecting plasmid DNA yield and purity

Cell density

Excessively high E. coli cell density is the most important factor resulting in the poor yield and purity of plasmid DNA. Cultures grown to an extremely high density (A₆₀₀ > 5) can overload both the mini- (1 to 3 mL) and midi-scale (~50 mL) purification systems. If high cell densities are obtained, you should process smaller culture volumes to ensure no deleterious effect on plasmid recovery and purity. The A₆₀₀ of an overnight culture of the E. coli strain TOP10 (transformed with a high-copy-number plasmid, >300 copies/cell) and grown in LB medium is approximately 2.5.

Note: LB is a nutritionally complex medium, primarily used for the growth of bacteria. Adjust salt levels as appropriate for the bacterial strain, culture conditions, and salt sensitivity of the antibiotic used.

Most techniques use a variation of alkaline lysis to lyse bacterial cells.

Technique	How it works	Advantages (+)/ Disadvantages (−)
Methods based on alcoholprecipitation (may includephenol/chloroform extraction,RNase treatment)	When cations (e.g., Na+ or K+) are present at a sufficient concentration to counteract the repulsion caused by the negative charges of the DNA backbone, DNA becomes insoluble in aqueous solutions and is precipitated. DNA is recovered by centrifugation.	+ Inexpensive − Plasmid DNA may be of low purity, particularly if phenol/chloroform extraction andRNase treatment is notincluded.
Methods using CsCl	In the CsCl method, supercoiled plasmid DNA is separated from linearized and “open circle” plasmid DNA due to different buoyant densities. Ethidium bromide intercalates into plasmid DNA, allowing visualization.	+ Extremely high purity + Good for scale-up − Low throughput − Requires access to ultracentrifuge and overnight centrifugation
Silica purification; usually in membrane or bead format	Plasmid DNA binds to silica in the presence of high concentration of chaotropes; DNA is eluted at low ionic strength.	+ DNA is eluted in low-ionicstrength buffer, ready for use + Amenable to increased throughput − May not remove sufficient endotoxins to obtain good transfection efficiencies with sensitive cell lines
Anion exchange chromatography; membrane or medium format	Negatively charged plasmid DNA binds to positively charged medium. DNA is eluted in high-ionic-strength buffer as anions displace bound plasmid DNA.	+ Few proteins bind, so protein removal is relatively complete + Effective removal of endotoxins − DNA is eluted in high salt and must be desalted before most applications
Gel filtration	Supercoiled plasmid DNA can be separated from contaminants by size selection.	+ Good for large scale − Low throughput
Amplification using Phi29 DNA polymerase	Phi29 DNA polymerase can be used to increase the amount of plasmid DNA. This is achieved by RCA using nonspecific primers.	+ Good when limited starting template material is available + Amenable to automation − Poor-quality input plasmid DNA may give poor-quality output

Growth conditions

Specific factors that affect culture growth, and ultimately the density of the culture, are listed below.

Inoculation

To inoculate growth medium for 1 to 3 mL cultures, use a fresh single transformed E. coli colony from an agar plate containing the appropriate antibiotics. For midi-prep scale, a small-scale starter culture (1 to 3 mL) is normally initiated as described above. After several hours of growth, this is used to inoculate a larger culture volume (> 50 mL).

Culture medium

When incubated for an equivalent period of time, cultures grown in enriched media such as 2× YT and TB tend to give cell densities that are significantly higher than those achieved with LB medium; therefore, the use of such enriched media when growing cultures for purifying plasmid DNA must be carefully considered in terms of cell density.

Aeration

Cultures should be well aerated during growth. When growing cultures in a 30 mL universal container, no more than 3 mL of medium should be used. Aeration will be poor if cultures are grown in multiwell plates or in 1.5 or 2.0 mL microcentrifuge tubes. Poor aeration will lead to poor culture growth, and subsequently to low yields of plasmid DNA. The problem of aeration is not as significant an issue when isolating plasmid DNA from midi-scale cultures, as these tend to be grown in large-volume flasks. However, a good “rule of thumb” is that the surface of the culture medium should be located near the widest part of the culture vessel to ensure that the maximum air to surface ratio is obtained.

Plasmid copy number

For a given length of incubation and a given culture medium, low-copy-number plasmids will give lower yields than high-copy-number plasmids.

Size of insert

In general, the larger the size of the insert, the lower the yield of plasmid DNA from a given culture medium.

Host strain

Strains that grow poorly or contain large amounts of nucleases or carbohydrates should be avoided. HB101 and its derivatives express endonuclease A (EndA+), which if not inactivated can digest plasmid DNA. These strains may also release carbohydrates that can inhibit restriction digests (5). E. coli strains DH5α and TOP10 are EndA- and therefore do not express an active nuclease. These strains facilitate the extraction of high-quality plasmid DNA.

Length of incubation

For cultures grown in an enriched medium (e.g., 2× YT or TB), the length of the incubation time should not exceed 12 h. Cultures in LB medium should be grown for at least 9 h to obtain sufficient cell mass for processing. Cultures (in any medium) should not be grown for more than 16 h, due to increased rates of cell death, which will affect the yield and quality of extracted plasmid DNA.

Tips for plasmid DNA preparation

Culture preparation

To obtain good aeration, use a sterile tube or flask with a volume of at least four times the volume of the culture. Choose a suitable tube or flask that facilitates the generation of the largest culture medium to air surface interface.

If purifying a high-molecular-weight or low-copy-number plasmid, process twice the recommended culture volume.

Do not process more than the recommended culture volume when the culture was grown in enriched media or when a high-copy-number plasmid was used. If using enriched media, measure the culture growth (A600) and dilute to ≤ 2.5. This is especially important to prevent the blockage of purification columns used in centrifuged-based plasmid miniprep extractions (1.5 to 3.0 mL culture volumes).

To thaw frozen, pelleted cells: Defrost thoroughly at room temperature for 5 to 15 min prior to use. Do not thaw for more than 15 min, as endogenous enzyme activity can affect plasmid yield and purity.

Alkaline lysis procedures

Resuspension: Cell resuspension can be achieved by gentle vortexing or by pipetting up and down. For mini-scale extractions, cells pelleted in microcentrifuge tubes can be resuspended by simply running the tube along the holes of an empty pipette tip box.

Note: Incomplete cell resuspension will result in reduced plasmid DNA recovery.

Cell lysis: Vigorous mixing will shear genomic DNA. These fragments will be copurified with the plasmid DNA. Do not vortex. Do not allow the lysis reaction to exceed 5 min. Alkaline lysis buffers contain NaOH, which will denature the plasmid DNA on prolonged incubation.

Neutralization: Do not shake or mix vigorously because genomic DNA will be sheared and may co-isolate with plasmid DNA; mix by gentle inversion.

Clarification: If supernatant is not clear after neutralization and centrifugation, transfer to a fresh tube and recentrifuge. Using a pipette, immediately transfer the clarified lysate to a fresh tube without disturbing the pellet. The small flecks of flocculent (precipitated KDS and denatured cellular debris) that remain on the surface of the clarified lysate should stick to the pipette.

Mini-scale purification using silica

Following neutralization and centrifugation, use a pipette to transfer the supernatant to the purification column containing a silica membrane. Use a pipette if possible or decant directly. It is important to avoid transferring any cellular debris to the column as this will affect the purity of the isolated plasmid DNA.

Elution: When eluting plasmid DNA from the column, be sure that all residual wash buffer has been removed. The presence of contaminating ethanol in the eluted plasmid DNA may affect downstream applications; therefore, care must be taken to ensure its complete removal.

Purification using anion exchange medium

Overdrying the plasmid DNA pellet (generated after isopropanol precipitation) will make the DNA difficult to dissolve. If the pellet was overdried, heat the TE buffer to 60 °C prior to addition, or add the buffer and gently roller mix overnight.

Preparation of plasmid DNA for in vitro transcription

If RNase is used during preparation of plasmid DNA, remove it completely by phenol/chloroform extraction or Proteinase K digestion. This is necessary because residual RNase will degrade RNA generated during in vitro transcription.