Boost your Miniprep
(December 1st, 2016) Imagine: it’s Friday afternoon and you want to do some maxipreps. Upon inspection, there’s not a single maxiprep column left in the kit box or in the lab’s storage room. US-American researchers might have found a solution to your problem.
Scientists from the University of North Carolina at Chapel Hill, led by Mark Peifer, have improved DNA miniprep protocols in such a way that they have been able to isolate maxiprep amounts of high copy number plasmid DNA at similar cost in under 30 minutes. Conventional maxipreps take two to five times as long. By using their method, which they have called ‘Miracle Preparation’ or ‘Miraprep’, your weekend can start that much earlier.
The scientists needed only 50 ml of bacterial culture per preparation: two to ten times less than for conventional maxipreps. The yield of high copy plasmid DNA was approximately 200 to 600 micrograms, and five spin columns were required per preparation. For their new protocol, the researchers used commercially available spin column miniprep kits from QIAGEN, Thermo Fisher (GeneJET) and Sigma (GenElute).
The method was as elegant as it was remarkable: adding one volume of ethanol after lysis and neutralisation, just before loading the DNA onto the silica miniprep column, did the trick. The authors suggest that the added ethanol precipitates the DNA, which is subsequently partially bound and partially retained by the column. Thus, the binding capacity of the column can be exceeded without loss of DNA.
The Mirapreps were not significantly contaminated with low molecular weight RNA. The isolated plasmids also survived an overnight incubation at 37 degrees Celsius unharmed and could be used for standard molecular biology procedures, such as DNA sequencing and the transfection of mammalian cells. The transfected heterologous genes were readily expressed.
The initial discovery was made by chance. “Some midi- and maxiprep kits use ethanol in their protocol, such that the samples are mixed with ethanol prior to loading on the column. When we ran out of midiprep columns, we loaded the ethanol-mix onto miniprep columns. Surprisingly, it worked better than with the original midiprep columns,” explained the first author Mira Pronobis, who was until recently a PhD student in Mark Peifer’s lab and who is now a post-doc in Kenneth Poss’ group at Duke University in Durham, North Carolina.
Asked what limitations her method might have, she said, “We have not determined the long-term stability of the DNA preparations, so this may be an issue. There may also be applications that require DNA of higher purity, such as embryo injections — however, our protocol worked very well for the standard applications we tested for the publication.” Mark Peifer’s group only occasionally investigates biotechnology tools. The lab’s main research focus is cell adhesion, cytoskeletal regulation and Wnt signaling in development and cancer. “However, if we discover something new in regards to common lab protocols, we will publish other improvements,” the professor commented.
For the DNA purification experts of QIAGEN, ‘Mirapreps’ are more or less old hat. Thorsten Singer, the company’s Director of R&D Sample Technologies Research, said that QIAGEN had explored a similar approach in 2002 in field tests under the name ‘AlphaPrep’, which even allowed the vacuum-assisted loading of the sample in one step. Especially with low copy plasmids, occasional contaminations with RNA were observed. “We therefore preferred an alternative binding solution which reduced contaminations with endotoxins and RNA even for low copy plasmid purifications, which require a higher input of bacteria. The improved version was launched a decade ago under the name ‘Plasmid Plus Kits’,” Singer explained.
Depending on the application of their plasmid DNA and the time available, scientists have the choice of which method they wish to use.