Plasmid Isolation using Quiagen QIAprep Spin Miniprep

 

Background

Growth of bacterial cultures

Plasmids are generally prepared from bacterial cultures grown in the presence of a selective agent such as an antibiotic. The yield and quality of plasmid DNA may depend on factors such as plasmid copy number, host strain, inoculation, antibiotic, and type of culture medium.

 

Plasmid copy number

Plasmids vary widely in their copy number per cell (Table-1), depending on their origin of replication (e.g., pMB1, ColE1, or pSC101) which determines whether they are under relaxed or stringent control; and depending on the size of the plasmid and its associated insert. Some plasmids, such as the pUC series and derivatives, have mutations which allow them to reach very high copy numbers within the bacterial cell. Plasmids based on pBR322 and cosmids are generally present in lower copy numbers. Very large plasmids and cosmids are often maintained at very low copy numbers per cell.

 

Table-1 Origin of Plasmids and Copy Number

 

Host strains

Most E. coli strains can be used successfully to isolate plasmid DNA, although the strain used to propagate a plasmid has an effect on the quality of the purified DNA. Host strains such as DH1, DH5a, and C600 give high-quality DNA. The slower growing strain XL1-Blue also yields DNA of very high-quality which works extremely well for sequencing. Strain HB101 and its derivatives, such as TG1 and the JM series, produce large amounts of carbohydrates, which are released during lysis and can inhibit enzyme activities if not completely removed (4). In addition, these strains have high levels of endonuclease activity which can reduce DNA quality. The methylation and growth characteristics of the strain should also be taken into account when selecting a host strain.  XL1-Blue and DH5a are highly recommended for reproducible and reliable results.

 

Inoculation

Bacterial cultures for plasmid preparation should always be grown from a single colony picked from a freshly streaked selective plate. Subculturing directly from glycerol stocks, agar stabs, and liquid cultures may lead to uneven plasmid yield or loss of the plasmid. Inoculation from plates that have been stored for a long time may also lead to loss or mutation of the plasmid.

The desired clone should be streaked from a glycerol stock onto a freshly prepared agar plate containing the appropriate selective agent so that single colonies can be isolated. This procedure should then be repeated to ensure that a single colony of an antibioticresistant clone can be picked. A single colony should be inoculated into 1–5 ml of media containing the appropriate selective agent, and grown with vigorous shaking for 12–16 hours. Growth for more than 16 hours is not recommended since cells begin to lyse and plasmid yields may be reduced.

 

Antibiotics

Antibiotic selection should be applied at all stages of growth. Many plasmids in use today do not contain the par locus which ensures that the plasmids segregate equally during cell division. Daughter cells that do not receive plasmids will replicate much faster than plasmid-containing cells in the absence of selective pressure, and can quickly take over the culture.

The stability of the selective agent should also be taken into account. Resistance to ampicillin, for example, is mediated by β-lactamase which is encoded by the plasmid-linked bla gene and which hydrolyzes ampicillin. Levels of ampicillin in the culture medium are thus continually depleted. This phenomenon is clearly demonstrated on ampicillin plates, where “satellite colonies” appear as the ampicillin is hydrolyzed in the vicinity of a growing colony. Ampicillin is also very sensitive to temperature, and when in solution should be stored frozen in single-use aliquots.

 

Table-2 Concentrations of Commonly Used Antibiotics

  

Culture media

Luria-Bertani (LB) broth (10 g Tryptone, 5 g Yeast extract, 10 g NaCl in 1L) is the recommended culture medium for use with QIAprep Kits, since richer broths such as TB (Terrific Broth) or 2x YT lead to extremely high cell densities, which can overload the purification system. If excess culture volume is used, alkaline lysis will be inefficient, the QIAprep membrane will be overloaded, and the performance of the system will be unsatisfactory. Furthermore, the excessive viscosity of the lysate will require vigorous mixing, which may result in shearing of bacterial genomic DNA and contamination of the plasmid DNA. Care must also be taken if strains are used which grow unusually fast or to very high cell densities. In such cases, doubling the volumes of Buffers P1, P2, and N3 may be beneficial. It is best to calculate culture cell density and adjust the volume accordingly.

Please note that a number of slightly different LB culture broths, containing different concentrations of NaCl, are in common use. Although different LB broths produce similar cell densities after overnight culture, plasmid yields can vary significantly.

 

Preparation of cell lysates

Bacteria are lysed under alkaline conditions. After harvesting and resuspension, the bacterial cells are lysed in NaOH/SDS (Buffer P2) in the presence of RNase A. SDS solubilizes the phospholipid and protein components of the cell membrane, leading to lysis and release of the cell contents while the alkaline conditions denature the chromosomal and plasmid DNAs, as well as proteins. The optimized lysis time allows maximum release of plasmid DNA without release of chromosomal DNA, while minimizing the exposure of the plasmid to denaturing conditions. Long exposure to alkaline conditions may cause the plasmid to become irreversibly denatured. This denatured form of the plasmid runs faster on agarose gels and is resistant to restriction enzyme digestion.

The lysate is neutralized and adjusted to high-salt binding conditions in one step by the addition of Buffer N3. The high salt concentration causes denatured proteins, chromosomal DNA, cellular debris, and SDS to precipitate, while the smaller plasmid DNA renatures correctly and stays in solution. It is important that the solution is thoroughly and gently mixed to ensure complete precipitation. To prevent contamination of plasmid DNA with chromosomal DNA, vigorous stirring and vortexing must be avoided during lysis. Separation of plasmid from chromosomal DNA is based on coprecipitation of the cell wall-bound chromosomal DNA with insoluble complexes containing salt, detergent, and protein. Plasmid DNA remains in the clear supernatant. Vigorous treatment during the lysis procedure will shear the bacterial chromosome, leaving free chromosomal DNA fragments in the supernatant. Since chromosomal fragments are chemically indistinguishable from plasmid DNA under the conditions used, the two species will not be separated on QIAprep membrane and will elute under the same low-salt conditions. Mixing during the lysis procedure must therefore be carried out by slow, gentle inversion of the tube.

 

Plasmid Isolation using Quiagen  QIAprep Spin Miniprep: Protocol

 

Preparation and clearing of bacterial lysate

The QIAprep miniprep procedure uses the modified alkaline lysis method of Birnboim and Doly. Bacteria are lysed under alkaline conditions, and the lysate is subsequently neutralized and adjusted to high-salt binding conditions in one step. After lysate clearing, the sample is ready for purification on the QIAprep silica membrane. Lysates are cleared by centrifugation.

 

1. Pick a single colony from a freshly streaked selective plate and inoculate a culture of 1–5 ml LB medium containing the appropriate selective antibiotic. Incubate for 12–16 h at 37°C with vigorous shaking.

Growth for more than 16 h is not recommended since cells begin to lyse and plasmid yields may be reduced. Use a tube or flask with a volume of at least 4 times the volume of the culture.

 

2. Harvest the bacterial cells by centrifugation at > 8000 rpm (6800 x g) in a conventional, table-top microcentrifuge for 3 min at room temperature (15–25°C).

The bacterial cells can also be harvested in 15 ml centrifuge tubes at 5400 x g for 10 min at 4°C.  Remove all traces of supernatant by inverting the open centrifuge tube until all medium has been drained.

 

Protocol

This protocol is designed for purification of up to 20 µg of high-copy plasmid DNA from 1–5 ml overnight cultures of E. coli in LB (Luria-Bertani) medium. Note: All protocol steps should be carried out at room temperature.

Procedure

1. Resuspend pelleted bacterial cells in 250 µl Buffer P1 and transfer to a microcentrifuge tube.

Ensure that RNase A has been added to Buffer P1. No cell clumps should be visible after resuspension of the pellet.

 

2. Add 250 µl Buffer P2 and mix thoroughly by inverting the tube 4–6 times.

Mix gently by inverting the tube. Do not vortex, as this will result in shearing of genomic DNA. If necessary, continue inverting the tube until the solution becomes viscous and slightly clear. Do not allow the lysis reaction to proceed for more than 5 min.

 

3. Add 350 µl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times.

To avoid localized precipitation, mix the solution thoroughly, immediately after addition of Buffer N3. Large culture volumes (e.g. 5 ml) may require inverting up to 10 times. The solution should become cloudy.

 

4. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.

 

5. Apply the supernatants from step 4 to the QIAprep spin column by decanting or pipetting.

 

6. Centrifuge for 30–60 s. Discard the flow-through.

 

7. Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 30–60 s.

 

8. Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer.

Important: Residual wash buffer will not be completely removed unless the flow-through is discarded before this additional centrifugation. Residual ethanol from Buffer PE may inhibit subsequent enzymatic reactions.

 

9. Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 µl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of each QIAprep spin column, let stand for 1 min, and centrifuge for 1 min.