by Caitlin Smith
So many labs use plasmid DNA—in applications such as PCR, transfection, cloning, and sequencing—that it’s no wonder there are many good purification kits available to speed its preparation, in the form of minipreps, midipreps, or maxipreps. Gone are the days-long protocols. And vendors are also constantly trying to increase the quality of their products to give you the best results in the lab. “When talking about improving plasmid purification, I think that the discussion is really about DNA yield and DNA quality,” says Michael Karberg, scientist at Zymo Research. “I believe that any procedure that minimizes hands-on time, processing time, and reagent-use will drive the future development of plasmid miniprep isolation kits.” Indeed, Jeff Briganti, strategic marketing manager at Promega, believes that an exciting recent development in plasmid DNA purification is the faster prep time. “It wasn't that long ago that preps took hours or even days,” says Briganti. “Now, you can get clean DNA in 10 minutes. The benefits are obvious. Researchers can spend more time on higher-value research rather than a mundane task like plasmid preps.”
Fast simplicity
A number of miniprep kits have reduced prep time to mere minutes. For example, Sigma-Aldrich’s GenElute™ Five-Minute Plasmid Miniprep Kits reduced prep time to five minutes. Similarly, Zymo’s Zyppy Plasmid Miniprep Kit is designed to save you time because its pellet-free protocol eliminates the conventional miniprep steps of cell-pelleting, removal of supernatant, and cell resuspension. “Instead, cell lysis and neutralization is performed directly in the culture growth medium,” says Karberg. “The procedure is also flexible so that our customers can still perform the classical centrifuge-based procedure if they wish.” Zymo’s kit gives average processing times of eight minutes for one to five samples, compared to tens of minutes in other systems.
Promega has also shaved its miniprep time down to 10 minutes (with midiprep 40 minutes, and maxiprep 60 minutes) with its new PureYield™ plasmid prep kits. Briganti claims that DNA purity distinguishes Promega’s kits from others. “There are other fast preps (some even faster), but none of those fast preps provide DNA that is as pure (based on A260/A280 ratios and endotoxin levels),” says Briganti. “The other new development is that customers are realizing that the concept of ‘endo-free’ preps was oversold. For most applications, low levels of endotoxin do not inhibit performance. Customers are no longer paying the high prices and wasting hours to purify ‘endo-free’ plasmid DNA.”
Quality, yield, and throughput
Obviously, high quality of DNA is important to most researchers who need to purify plasmid DNA. It is essential, however, “for applications such as transfection, electroporation, and PCR, where endotoxins, PCR inhibitors, and salts can dramatically affect the success of an experiment,” says Karberg. But Karberg adds that low amounts of the most common contaminants in plasmid minipreps—chromosomal DNA, nicked or relaxed plasmid DNA, and RNA—don’t interfere with most subsequent applications, such as PCR, sequencing, cloning, and transformation.
Yield is another measure of difference between purification kits, but Karberg points out that there are ways to remedy a yield that is lower than expected. “In my opinion, DNA yield is important for ultra-low copy number plasmids, plasmid isolation from Gram-positive or exotic bacteria, and for the few researchers who require milligram quantities of DNA,” says Karberg. “These challenges are mostly overcome by simply increasing the amount of culture that is processed, or by using fermentation equipment, if available to the researcher. Most plasmid DNA miniprep kits supply sufficient DNA for the most common lab applications of sequencing, cloning, transformation/transfection, and PCR.” For processing many samples in parallel, several companies offer high-throughput formats for plasmid preparation. For example, USB (now owned by Affymetrix) offers their PrepEase® Plasmid 96-well Plate Kit, which uses classical alkaline lysis followed by vacuum filtration (centrifugation is optional) with filter plates and plasmid binding plates.
Plasmid DNA purification has seen strides in speed, quality, yield, and convenience. Is there much room left for improvement? “The biggest challenge is that nearly all kits are utilizing the same basic underlying technology (silica),” says Briganti. “Periodically, we see small incremental improvements. But it is unlikely that we'll see any major leaps using silica going forward. On the other hand, do we need a major leap at this point? How much can you improve on a 10-minute process that results in highly pure DNA?”