Deconstructing the Plasmid Prep – Tips For Optimization
One of the most commonly performed laboratory tasks across many disciplines of molecular biology is the purification of plasmid DNA from bacteria. The ubiquity of this process has led to the popularization of commercially available plasmid extraction kits taking the place of the standard cesium-chloride gradient purification. While plasmid extraction kits are available from many different research suppliers, the process is mostly the same across all of them. In short, bacterial cells are grown in liquid culture, lysed by an alkaline buffer, then the resulting plasmid DNA is purified by a series of column elutions and buffer washes. While these commercially available kits simplify the plasmid extraction process with their premade buffers and simple instructions, it is important to note the purpose of each step to properly assess what may be going wrong should any problems arise. This article will break down what goes on during each step of the plasmid extraction process and where any deviations may impact the final yield.
Before You Begin
While the standard extraction protocol applies to most constructs, some changes may be necessary to obtain high yields from plasmids with certain characteristics. It is important to determine these characteristics (plasmid size, copy number, host strain) before attempting an extraction. Standard extraction kits are capable of purifying plasmids up to 150 kb in size, but any construct larger than 50 kb should use an elution buffer heated to about 65kb. These larger constructs are more likely to stick to the filter during the elution step, so using a warmed elution buffer helps to release them into the final eluate. For any plasmids that exceed the maximum size limit of the kit being used, special kits designed for large plasmid must be used. The copy number of the plasmid will dictate its concentration in the cell lysate. For plasmids with very high copy numbers, a reduced volume of liquid cultures should be used to avoid overloading the spin columns. Likewise, plasmids with a very low copy number should increase the volume of liquid cultures to compensate for the low concentration. Lastly, the host strain used can also affect the final yield. Ideally, strains with low endonuclease activity should be used to prevent the degradation of produced DNA. Due to the myriad of strains to choose from, it is prudent to consult the kit’s guidebook to obtain recommendations for optimal host strains.
Step One – Growth of Liquid Cultures
The purpose of this step is to grow bacterial cells transformed with the plasmid of interest for purification during the following steps. To identify an ideal host strain, consult the previous section of this article or the handbook provided by the extraction kit. To begin, transformed cells should be streaked on a plate with an antibiotic corresponding to the antibiotic resistance gene conferred by the construct. This will allow for the selection of single colonies that contain the plasmid of interest. Single colonies should then be picked and used to inoculate tubes of LB medium. These liquid cultures should be grown to the end of the logarithmic phase of growth, ideally not well into the stationary phase to avoid cell death and subsequent release of endonucleases. Growth should be monitored using OD600 measurements, and typically takes anywhere from 12-16 hours. Key factors to keep in mind here are the selection of colonies and the density of the final liquid culture. If plates with no antibiotic are used, properly transformed cells cannot properly be selected for and will result in a greatly reduced yield. The same can be said if the liquid cultures are not grown to a proper cell density, or if the liquid cultures are overgrown and mass cell death has occurred.
Step Two – Resuspension and Lysis of Liquid Cultures
After the bacterial cultures have been grown, they are pelleted in a centrifuge and resuspended in the kit’s resuspension buffer, which equilibrates cells to the extraction buffer. Next, an alkaline buffer is added to lyse the cells. This buffer is usually composed of a strong base such as sodium hydroxide, a detergent such as sodium dodecyl sulfate, and an RNase to break down any free RNA. The detergent fully solubilizes the cell membrane, which exposes the cellular contents to the alkaline buffer which denatures large macromolecules such as chromosomal DNA, plasmid DNA, and proteins. Step two is the step that is most sensitive to time, as the lysis process needs to be neutralized at the time point outlined in the kit’s instructions. Neutralization that occurs too early will result in incomplete solubilization of cell membranes, preventing the maximum amount of plasmid to be released for later purification. Neutralization that occurs too late will result in plasmid DNA being damaged by the alkaline buffer. For best results, it is critical to end the lysis step precisely when the kit specifies. Step two is also highly sensitive to the conditions in which the reactions are handled. During lysis, chromosomal DNA is highly susceptible to shearing so reactions should be handled as gently as possible. If reactions are mixed too vigorously, i.e. by vortexing, chromosomal fragments will be unable to be removed during the purification steps and will contaminate the final plasmid sample.
Step Three – Neutralization of Lysis Reactions
After the lysis reaction is complete, an acidic buffer is added to neutralize the reaction. As a result, chromosomal DNA, proteins, and any remaining detergent precipitate out of solution. Plasmids are small enough to remain in solution and are capable of renaturing now that all the detergent is trapped in salt-detergent complexes. Since plasmids will not bind to centrifuge column resin if they are still denatured, the solution must be thoroughly mixed to ensure the complete precipitation of the detergent. As with step two, mixing should be done gently by inverting the tube to prevent any shearing of chromosomal DNA. The precipitates can then be removed from the neutralized mixture by centrifugation.
Step Four – Elution of Pure Plasmid DNA
After step three is complete, the cleared lysate can then be loaded onto a centrifugal column provided by the kit. An initial spin is done to bind the plasmid DNA to the column’s resin. The salt and pH conditions of the lysate ensure that plasmids selectively bind to the resin whereas any soluble proteins or other contaminants flow through. A medium salt buffer is added to the column and allowed to flow through during another spin which removes any contaminants that may have bound to the resin. Finally, a high salt buffer is used to elute bound plasmids from the resin. If the elution buffer has been used for previous extractions, it is recommended that the elution buffer be autoclaved before use to remove any nucleases that may have contaminated it.
In conclusion, the user-friendly plasmid extraction kits greatly simplify a common lab task. While the premade buffers make the process easy to follow, this can lead to a disconnect between researchers and the rationale of each step. A summary of each step is as follows. The first step grows transformed bacterial cells in liquid culture and errors can arise from incorrect growth density or choosing of an improper host strain. The second step is lysis of the resuspended cells and errors can arise from incubating too long or too little in the alkaline buffer. The third step is the neutralization of the alkaline buffer and errors can arise from overly vigorous mixing. Lastly, the fourth step is the elution of pure plasmid DNA and errors can arise from RNase contaminated elution buffer.
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