Plasmid Purification

Plasmid purification is a technique used to isolate and purify plasmid DNA from genomic DNA, proteins, ribosomes, and the bacterial cell wall. A plasmid is a small, circular, double-stranded DNA that is used as a carrier of specific DNA molecules.

The purification of plasmid DNA from bacterial cells is an important step in the cloning workflow.  During plasmid purification, bacterial cells are lysed, freeing DNA and other cellular components from the cell wall. Cellular components are then removed, and the DNA-containing lysate is processed to further remove contaminants separate the plasmid DNA from the genomic DNA.

Alkaline lysis was first described by Birnboim and Doly in 1979 (Nucleic Acids Res. 7, 1513-1523) and has, with a few modifications, been the preferred method for plasmid DNA extraction from bacteria ever since. The easiest way to describe how alkaline lysis works is to go through the procedure and explain each step, so here goes.

Cell Growth and Harvesting

The procedure starts with the growth of the bacterial cell culture harboring your plasmid. When sufficient growth has been achieved, the cells are pelleted by centrifugation to remove them from the growth medium.

Re-suspension

The pellet is then re-suspended in a solution (normally called solution I, or similar in the kits) containing Tris, EDTA, glucose and RNase A. Divalent cations (Mg2+, Ca2+) are essential for DNase activity and the integrity of the bacterial cell wall. EDTA chelates divalent cations in the solution preventing DNases from damaging the plasmid and also helps by destabilizing the cell wall. Glucose maintains the osmotic pressure so the cells don’t burst and RNase A is included to degrade cellular RNA when the cells are lysed.

Lysis

The lysis buffer (aka solution 2) contains sodium hydroxide (NaOH) and the detergent Sodium Dodecyl (lauryl) Sulfate (SDS). SDS is there to solubilize the cell membrane. NaOH helps to break down the cell wall, but more importantly it disrupts the hydrogen bonding between the DNA bases, converting the double-stranded DNA (dsDNA) in the cell, including the genomic DNA (gDNA) and your plasmid, to single stranded DNA (ssDNA). This process is called denaturation and is central part of the procedure, which is why it’s called alkaline lysis. SDS also denatures most of the proteins in the cells, which helps with the separation of the proteins from the plasmid later in the process.

It is important during this step to make sure that the re-suspension and lysis buffers are well mixed, although not too vigorously (see below). Check out my article on 5 tips on vector preparation for gene cloning for more information and tips. Also remember that SDS and NaOH are pretty nasty so it’s advisable to wear gloves and eye protection when performing alkaline lysis.

Neutralization

Addition of potassium acetate (solution 3) returns decreases the alkalinity of the mixture. Under these conditions the hydrogen bonding between the bases of the single stranded DNA can be re-established, so the ssDNA can re-nature to dsDNA. This is the selective part. While it is easy for the the small circular plasmid DNA to re-nature it is impossible to properly anneal those huge gDNA stretches. This is why it’s important to be gentle during the lysis step because vigorous mixing or vortexing will shear the gDNA producing shorter stretches that can re-anneal and contaminate your plasmid prep.

While the double-stranded plasmid can dissolve easily in solution, the single stranded genomic DNA, the SDS and the denatured cellular proteins stick together through hydrophobic interactions to form a white precipitate. The precipitate can easily be separated from the plasmid DNA solution by centrifugation.

5. Cleaning and concentration

Now your plasmid DNA has been separated from the majority of the cell debris but is in a solution containing lots of salt, EDTA, RNase and residual cellular proteins and debris, so it’s not much use for downstream applications. The next step is to clean up the solution and concentrate the plasmid DNA.

There are several ways to do this including phenol/chloroform extraction followed by ethanol precipitation and affinity chromotography-based methods using a support that preferentially binds to the plasmid DNA under certain conditions of salt or pH, but releases it under other conditions. The most common methods are detailed in my article on 5 ways to clean up a DNA sample.

Written by Dr Nick Oswald

Conveniently, there are several ways to clean up DNA. Here are five of them:

Phenol-Chloroform Extraction

Phenol chloroform extraction (see Kirby, 1957), normally followed by ethanol precipitation, is the traditional method to remove protein from a DNA sample. In this procedure, the DNA solution is mixed with phenol and chloroform. The water-soluble DNA partitions into the aqueous phase, while the proteins denature in the presence of organic solvents, thus staying in the organic phase. The aqueous phase containing the protein-free DNA is then ready for collection.

Advantages: A cheap and effective way to remove proteins from DNA solutions.

Disadvantages: Slow compared to most modern methods. There is a risk of phenol/chloroform carry-over into the final sample (which could inhibit downstream enzymatic reactions). Chloroform and phenol are both hazardous chemicals.

Ethanol Precipitation

Ethanol precipitation is a popular method for desalting and concentrating DNA. Monovalent cations (0.1 to 0.5 M, normally in the form of the acetate salt of sodium) are added to the DNA, along with ethanol to a final concentration of 70%. Ethanol changes the DNA structure so that the DNA molecules aggregate and precipitate out of solution (see Eickbush and Moudrianakis, 1978). Most salts and small organic molecules are soluble in 70% ethanol, leaving the precipitated DNA ready for separation by centrifugation.

Advantages: A cheap and effective way to desalt and concentrate DNA.

Disadvantages: Time consuming. You risk carrying ethanol over into the final sample.

3. Silica Column-Based Kits

Column-based kits offer a convenient approach to DNA cleanup. The principle is that chaotropic salts are added to the sample to denature the DNA by disrupting its hydrogen bonds. Under these conditions, the DNA will selectively bind to the silica resin in the column, facilitating its separation from the rest of the sample. After washing, the DNA is eluted from the column with a low salt solution that allows renaturing, causing the DNA to lose affinity for the silica. Most commercial suppliers offer kits based on this technology, with a range of kits available for DNA cleanup after agarose gel extraction, enzymatic reactions and PCR, to name a few.

Advantages: Convenient, relatively fast and the user can process a large number of samples using the vacuum manifold option.

Disadvantages: May be costly. Some kits give low yields (as low as 25%). Chaotropic salt carryover may be an issue.

Note: Some suppliers are now offering improved silica column kits, where sample elution takes place in a very small volume to yield high DNA concentrations.

4.  Anion Exchange

Anion exchange DNA purification methods usually use positively charged DEAE functionalized resins to bind the negatively charged DNA phosphate backbone. Using specific salt and pH conditions, DNA in the sample binds the resin, and stringent washing steps remove contaminants (e.g. protein, cellular debris). The remaining DNA can then be selectively eluted from the resin.

Advantages: high purity DNA for downstream applications e.g. transfection and DNA sequencing.

Disadvantages: resins can be expensive.

5. Magnetic Beads

This approach uses magnetic beads that conditionally bind DNA in a pH-dependent manner, allowing you to separate the DNA from the rest of the sample by simply controlling pH. There are a few magnetic bead kits on the market. The magnetic beads are positively charged and bind DNA at low pH, but at high pH they become negatively charged, thus releasing the DNA.

Advantages: Fast, no chaotrophic salts or organic washing solutions required. Ideal for automation of high throughput processing, since there is no need for centrifugation and other time consuming processing steps. Note there are also magnetic bead kits that selectively bind DNA depending on salt concentration.

Disadvantages: The initial cost in purchasing the magnets is reasonably high. This procedure can be tricky when handling multiple samples, but this is not an issue if you have access to an automated workflow.

Summarized method--3

For this procedure a lab coat, disposable gloves and protective goggles should be worn.

The bacterial cultures, which are transformed with the desired plasmid DNA, should be grown overnight in growth media with appropriate antibiotic in a 37°C shaking incubator.

The next day, the bacteria culture is pelleted by centrifugation and the supernatant is removed. The remaining pellet is resuspended in lysis buffer.

Once resuspended, bacteria can be transferred to a smaller tube where lysis buffer is added. Lysis buffer has a high pH and contains detergents, such as sodium dodecyl sulfate, which disrupt the bacterial membranes, thereby lysing the bacteria. The solution will turn cloudy with the addition of the lysis buffer and after mixing the solution becomes more clear. The mixture should not be vortexed due to the possibility of shearing, or breaking apart, genomic DNA, which could then contaminate plasmid DNA purification.

Then, neutralization buffer is added to neutralize the alkaline conditions and lower the pH. With gentle mixing genomic DNA as well as any proteins bound to it will precipitate, while plasmid DNA will stay in solution. Once again avoid vortexing, so as your plasmids are free of genomic DNA contamination.

The mixture is then centrifuged and the genomic DNA/protein precipitate is pelleted. The supernatant contains the plasmid DNA as well as soluble proteins.

This supernatant is placed onto the column by decanting, a fancy name for pouring it off, or pipetting. The pellet can be discarded.

Next, high salt washing buffer passes through the column while the DNA remains bound to the silica. Repeated washing steps with high salt buffer removes endonucleases, RNA, proteins, dyes, and low-molecular weight impurities. The flow-through of the wash steps should be discarded. After the final washing step make sure the filter is completely dry without any buffer remaining. The plasmid DNA is still located on the filter.

The plasmid DNA is eluted with sterile water or an elution buffer. The DNA is readily available for immediate use in a wide range of applications.

There are a number of ways to verify the purity of plasmids after purification. A spectrometer can be used to compare absorbance at different wavelengths to determine the concentration of plasmid DNA. An agarose gel analysis of the purified plasmid can determine if the plasmid is the correct size and there are no contaminants. This step ensures there is no genomic DNA contamination and the plasmid was not modified in bacteria.

Plasmid Purification

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