Plasmid Isolation 22nd October 2021 – Tags:

Introduction

Plasmid isolation consists of five steps: cell lysis, debris removal, nucleic acid precipitation, nucleic acid isolation and resuspension.

A plasmid preparation is a method of extracting and purifying plasmid DNA. Many methods have been developed to purify plasmid DNA from bacteria.

These methods invariably involve three steps:

  • Growth of the bacterial culture
  • Collection and lysis of bacteria
  • Plasmid DNA purification

The term “plasmid” was coined by Joshua Lederberg in 1952, and plasmid isolation is based on this term. Plasmids are elements genetic extrachromosomes present in most species of Archae, Eukarya and Eubacteria.

Plasmids are circular, double-stranded DNA molecules that are distinct from the chromosomal DNA of cells.

The structure and function of a bacterial cell are directed by the genetic material contained in chromosomal DNA. In some cases, plasmids are usually not essential for the survival of the host bacterium.

Plasmids contribute to the genetic diversity and plasticity of bacteria. They encode functions that may not be specified by bacterial chromosomal DNA.

Plasmids specify traits that allow the host to persist in otherwise lethal or growth-restrictive environments. For example, antibiotic resistance and protein expression. Antibiotic resistance genes are often encoded by the plasmid, allowing the bacterium to persist in an environment containing antibiotics.

This gives the bacteria a competitive advantage over antibiotic-sensitive species. In addition, as a tool, plasmids can be modified to express the protein of interest (e.g. the production of human insulin using recombinant DNA technology).

Plasmids have served as invaluable model systems for the study of processes such as DNA replication, segregation, conjugation and evolution. Plasmids have been central to modern recombinant DNA technology as a tool for gene cloning and as a vehicle for gene expression.

plasmid isolation cell lysis

Plasmid characteristics

The plasmids present in bacteria differ in their physical properties:

  • the size (kbp)
  • the geometry
  • the number of copies

Plasmid size

Plasmids range in size from 1 kbp (kilo base pair) to 1000 (kilo base pair) megaplasmids that have many hundreds of base pairs.

Plasmid geometry

Although most plasmids have a circular geometry, there are now many examples of plasmids that are linear in a variety of bacteria.

Plasmid DNA can appear in one of five conformations: Nicked open circular DNA, which has one strand cut; relaxed circular DNA, which is fully intact with both strands uncut, but has been enzymatically relaxed; linear DNA, which has free ends; supercoiled DNA, which is fully intact with both strands uncut; and denatured supercoiled DNA, which is like supercoiled DNA, but has unpaired regions that make it slightly less compact.

Plasmid copy numbers

Copy number refers to the average or expected number of copies per host cell. Plasmids have a low, medium or high copy number. Knowing which category the plasmid belongs to is very important when starting an experiment.

If you work with a low copy number plasmid that is associated with a low yield and therefore you might need to set up more cultures. On the other hand, if low yield is obtained with a high copy number plasmid, troubleshooting is required.

In bacteria with high copy number plasmids, during cell division plasmids segregate randomly into daughter cells, whereas in low copy number bacteria, during cell division and partitioning plasmids divide equally into daughter cells.

An advantage of high copy number is the increased stability of the plasmid when random partitioning (i.e. partitioning of plasmids into daughter cells) occurs during cell division.

Aislamiento de plásmidos

Isolation of plasmid DNA from bacteria is a crucial technique in molecular biology and is an essential step in many procedures such as cloning, DNA sequencing, transfection and gene therapy. These manipulations require the isolation of high purity plasmid DNA. Purified plasmid DNA can be used immediately in all molecular biology procedures such as restriction enzyme digestion, cloning, PCR, transfection, in vitro translation, blotting and sequencing.

Alkaline lysis is a method used in molecular biology to isolate plasmid DNA or other cellular components, such as proteins, by cleaving the cells. Bacteria containing the plasmid of interest are first cultured and then lysed with an alkaline lysis buffer consisting of a sodium dodecyl sulphate (SDS) detergent and a strong sodium hydroxide base. The detergent scales the phospholipid bilayer of the membrane and the alkali denatures the proteins involved in maintaining the cell membrane structure. Through a series of steps involving shaking, precipitation, centrifugation and removal of the supernatant, cell debris is removed and the plasmid is isolated and purified.

Principle

The purification of plasmid DNA from bacterial DNA is based on differential denaturation of chromosomal and plasmid DNA by alkaline lysis to separate the two.

The basic steps of plasmid isolation are disruption of the cell structure to create a lysate, separation of the plasmid from the chromosomal DNA, cell debris and other insoluble material. Bacteria are lysed with a lysis buffer containing sodium dodecyl sulphate (SDS) and sodium hydroxide. During this step, most of the cells are disrupted, chromosomal and plasmid DNA is denatured and the resulting lysate is cleaned by centrifugation, filtration or magnetic stripping. Subsequent neutralisation with potassium acetate allows only the covalently locked plasmid DNA to rejoin and remain solubilised. Most of the chromosomal DNA and proteins precipitate in a complex formed with potassium and SDS, which is removed by centrifugation.

The bacteria are resuspended in resuspension buffer (50mM Tris-Cl, 10 mM EDTA, 100 µg/ ml RNase A, pH 8.0) and then treated with 1% SDS (w/v) / alkaline lysis buffer (200mM NaOH) to release the plasmid DNA from the E. coli host cells.

Neutralisation buffer (3.0 M potassium acetate, pH 5.0) neutralises the resulting lysate and creates conditions suitable for plasmid DNA binding to the silica membrane column. The precipitated protein, genomic DNA and cell debris are separated by a centrifugation step and the supernatant is loaded onto a column.

Contamination such as salts, metabolites and soluble macromolecular cellular components are removed by simple washing with ethanolic wash buffer (1.0 M NaCl, 50mM MOPS, pH 7.0, isopropanol (v/v) 15 %).

Pure plasmid DNA is finally eluted under low ionic strength conditions with slightly alkaline buffer (5 mM Tris/HCl, pH 8.5).

The media

The yield and quality of plasmid DNA is highly dependent on the type of culture medium used. Most plasmid purifications are optimised with cultures on standard Luria Bertani (LB) medium.

To prepare LB medium, dissolve 10 g tryptone, 5 g yeast extract and 10 g NaCl in 800 ml distilled water. Adjust the pH to 7,0 with 1 N NaOH. Adjust the volume to 1 litre by adding distilled water and autoclave.

The cell culture should be incubated at 37 °C with constant shaking (200-250 rpm) preferably for 12 to 16 hours overnight. Generally, an OD of 3-6 can be achieved. Alternatively, rich media such as 2 x YT (yeast/tryptone), TB (terrifying broth) or CircleGrow can be used.

Care should also be taken, as overgrowth of a culture can lead to a higher percentage of dead or starved cells and the resulting plasmid DNA could be partially degraded or contaminated with chromosomal DNA. To find the optimal culture conditions, culture medium and incubation times have to be optimised for each host strain/plasmid construct combination individually.

Lisado y neutralización

Lysis formulas may vary depending on whether DNA/RNA/plasmid extraction is desired. All bacterial lysis methods will produce plasmid solutions contaminated with chromosomal DNA and RNA. Centrifugation removes the vast majority of chromosomal DNA (it will form a pellet, while plasmid DNA remains soluble), and RNase treatment will remove contaminating RNA.

In general, lysis buffers contain a high concentration of chaotropic salts. Chaotropes have two important functions in nucleic acid extraction. First, they destabilise hydrogen bonds, Van der Waals forces and hydrophobic interactions, leading to destabilisation of proteins, including nucleases. Secondly, they disrupt the association of nucleic acids with water, thus providing optimal conditions for their transfer to silica.

Plasmids are separated and removed from the bacterial cell by resuspending 1-5 mL of culture in resuspension buffer (50 mM Tris-Cl, 10 mM EDTA, 100 µg/ ml RNase A, pH 8.0) and pelleting the cells in a microcentrifuge at 11000 x g for 30 s. The cells are then lysed by adding 250 µg/ ml of culture buffer (50 mM Tris-Cl, 10 mM EDTA, 100 µg/ ml RNase A, pH 8.0).

Lysing is achieved by adding 250 µl lysis buffer with neutralisation buffer, as it aids complete precipitation of SDS, proteins and genomic DNA. Incomplete neutralisation leads to a reduction in yield. However, the released plasmid DNA is very vulnerable at this point and shaking too much or too hard will damage the DNA.

Fixing and washing on the silica membrane

After centrifugation of the lysate through the silica membrane, the desired nucleic acids should be bound to the column and impurities, such as proteins and polysaccharides, should be in the flow.

In the case of plant samples, they are likely to contain polysaccharides and pigments, while in the case of blood samples, the membrane may have a slightly brown or yellow colour. Washing steps will remove these impurities. There are usually two washing steps, although this varies depending on the type of sample.

The first wash usually involves a low concentration of chaotropic salts to remove residual proteins and pigments. This is always followed by an ethanol wash to remove salts. The columns contain a silica resin that selectively binds DNA/RNA. The DNA of interest can be isolated by virtue of its ability to bind to silica in the presence of high concentrations of chaotropic salts. These salts are removed with an alcohol-based wash and the DNA is eluted using a low ionic strength solution such as TE buffer or water.

DNA binding to silica appears to be driven by dehydration and the formation of hydrogen bonds, which compete against the weak electrostatic repulsion. Therefore, a high salt concentration will help drive DNA adsorption on silica, and a low concentration will release the DNA.

Elución

The volume of elution buffer and the method can be adapted to the downstream application to achieve a higher yield and/or concentration than the standard method. Elution buffer is used to wash out unbound proteins at the beginning and, at a higher concentration, releases the desired protein from the ligand. It is important that the elution buffer acts quickly without changing the function or activity of the desired protein. For maximum DNA elution, allow the buffer to sit on the membrane for a few minutes before centrifugation. Elution buffer AE (5 mM Tris/HCl, pH 8.5) can be replaced by TE buffer or water. It is preferable to use a weakly buffered, slightly alkaline buffer that does not contain EDTA, especially if the plasmid DNA is intended for sequencing reactions.

Analytical analysis of the gel

It is recommended to remove and save aliquots during the purification procedure.

If the plasmid DNA is of low yield or poor quality, samples can be analysed by agarose gel electrophoresis to determine at which stage of the purification procedure the problem occurred.

Procedure

Harvest Bacterial and Resuspended Cells

1. Pick a single colony from a freshly spread selective plate and inoculate a 2-5 ml starter culture of LB medium containing the appropriate selective antibiotic. Incubate for approximately 8 hours at 37°C with vigorous shaking (approximately 300 rpm).

2. Dilute the starter culture 1/500 to 1/1000 in 3 ml of selective LB medium. Culture at 37°C for 12-16 h with vigorous shaking (approx. 300 rpm).

3. Collect bacterial cells by centrifugation at 6000 x g for 15 min and remove as much of the supernatant as possible. Resuspend the bacterial pellet in 0.1-0.5 ml resuspension buffer (50 mM Tris-Cl, 10 mM EDTA, 100 µg/ ml RNase A, pH 8.0). Bacteria should be completely resuspended by vortexing or pipetting up and down until no cell clumps remain.

cel lysis

4. Add 0.25 ml lysis buffer, mix well by vigorously inverting the sealed tube 4-6 times, and incubate at room temperature (15-25°C) for 5 minutes. Do not vortex, as this will cause shearing of the genomic DNA. The lysate should have a viscous appearance. Do not allow the lysis reaction to last longer than 5 minutes.

Neutralisation

5. Add 0.3 ml of neutralisation buffer, mix immediately and thoroughly by vigorously inverting 4-6 times, and incubate on ice for 5 minutes. Precipitation is enhanced if cold neutralisation buffer is used and incubated on ice. After addition of the neutralisation buffer, a white, fluffy material forms and the lysate becomes less viscous. The precipitated material contains genomic DNA, proteins, cell debris and KDS. The lysate must be mixed thoroughly to ensure uniform precipitation of potassium dodecyl sulphate. If the mixture still appears viscous, further mixing is necessary to completely neutralise the solution. A homogeneous, colourless suspension indicates that the SDS has indeed precipitated.

Load the lysate onto the column

6. Before loading the column, carefully remove the supernatant and transfer it to a collection tube containing the column and centrifuge at 13,000 rpm for 1 minute.
7. Discard the flow-through liquid and quickly remove the supernatant containing the plasmid DNA. After centrifugation, the supernatant should be clear.
8. If the supernatant is not clear, a second, shorter centrifugation should be performed to avoid application of suspended or particulate material to the column. Suspended material (which makes the sample appear cloudy) will clog the column and reduce or eliminate flow.

Binding and washing

9. Add 0,7 ml of wash buffer to the column placed in the collection tube and centrifuge for 10 minutes at 13000 rpm for 1 minute. Equilibrate by applying 1 ml of equilibration buffer (750 mM NaCl, 50 Mm MOPS, ph 7.0, 15 % isopropanol) and allow the column to empty by gravity flow. The flow of the buffer will start automatically by the reduction of the surface tension due to the presence of detergent in the equilibration buffer.
10. Apply the supernatant from step 6 to the column and allow it to enter the resin by gravity flow.

Plasmid elution

11. Elute the DNA with 0.8 ml elution buffer (1.23 M NAcL, 50 mm Tris-Cl, pH 8.5, 15%v isopropanol) Collect the eluate in a 1.5 ml or 2 ml microcentrifuge tube.
12. Precipitate the DNA by adding 0,7 volumes (0,56 ml per 0,8 ml elution volume) of isopropanol at room temperature to the eluted DNA. Mix and centrifuge immediately at ≥10,000 rpm for 30 min in a microcentrifuge. Carefully decant the supernatant. All solutions should be at room temperature to minimise salt precipitation.
13. Wash the DNA pellet with 1 ml of 70% ethanol and centrifuge at 10,000 rpm for 10 min.
14. Carefully decant the supernatant without disturbing the pellet.
15. 70% ethanol removes the precipitated salt and replaces isopropanol with the more volatile ethanol, which facilitates DNA redissolution.
16. Air dry the pellet for 5-10 minutes and redissolve the DNA in an appropriate volume of buffer (e.g. TE buffer, pH 8.0, or 10mM Tris-Cl, pH 8.5). Redissolve the DNA pellet by rinsing the walls to recover all DNA.

Determining performance

To determine the yield, the DNA concentration should be determined both by UV spectrophotometry at 260 nm and by quantitative analysis on an agarose gel. To quantify the nucleic acid concentration, dilute the plasmid DNA 1 : 100 or 1 : 50 (depending on the plasmid copy number) in TE buffer and measure the absorbance (optical density) at 260 nm (A260) and 280 nm (A280). Use the TE buffer as a blank. This measurement allows direct calculation of the nucleic acid concentration using the formula

[DNA] (μg/mL) = A260 × Dilution factor × 50

where 50 is the extinction coefficient of DNA. The ratio A260/A280 provides a reasonable estimate of the purity of the preparation.

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