Product Survey: Plasmid extraction kits

Don’t Waste your Money
by Harald Zähringer, Labtimes 04/2017

The majority of plasmid extraction kits is based on pretty expensive silica spin columns. However, cheap salting-out kits are still an option – even for sensitive, downstream applications, such as sequencing.

Extracting plasmid DNA is one of the most fundamental tasks in life science labs, no matter what the specific research interests are. Cloning, restriction digests, recombinant DNA expression, preparation of microbial DNA for sequencing, transfections of plants via A. tumefaciens – countless molecular biology techniques require the isolation of plasmid DNA.

All plasmid extraction kits are based on similar buffers and protocols.

Breaking down the cell walls

Over the years, molecular biologists have come up with different plasmid isolation techniques, which may be roughly classified according to the initial cell disruption step into four categories: alkaline extraction, detergent-based methods, organic extraction techniques and physical treatments.

Alkaline extraction, which is the most popular plasmid isolation technique, is based on the disintegration of the cell wall with NaOH in the presence of SDS or other detergents, such as Triton X-100 or Brij-58; followed by alcohol precipitation of released plasmid DNA.

Detergent-based techniques rely on cell disruption with SDS alone or in combination with lysozyme and pronase. Organic procedures on the other hand, take advantage of phenol or phenol/chloroform mixtures, sometimes together with detergents or lysozyme, to extract plasmid DNA. Similarly, physical treatments, such as boiling, are also frequently performed in the presence of lysozyme and detergents.

Clever combination

These ages-old standard techniques are still widely in use. Many researchers, however, prefer fast, simple to use and phenol/chloroform-free commercial plasmid isolation kits, based on silica spin columns or silica-covered magnetic beads for plasmid extraction. Silica spin kits cleverly combine traditional alkaline cell lysis with pH-dependent binding of plasmids to DNA-adsorbing materials like silica.

Since adsorbance of DNA to silica surfaces is supported by low pH and chaotropic substances, such as GuSCN or NaI, the alkaline lysate is neutralised after NaOH/SDS lysis with a chaotropic buffer before loading on the spin column in the next step of the protocol.

Simple but efficient

Bound DNA is subsequently eluted from the silica membrane after a mandatory washing step with an alkaline low salt buffer – a simple, albeit very efficient purification process that yields very pure plasmid DNA and pours a lot of money into the coffers of kit manufacturers.

Spin column kits are not exactly a bargain. Costs per DNA sample preparation are considerably higher compared to traditional techniques. But are spin column based kits always necessary and worth the money?

To answer this question, Ulrich Nübel’s group at the Leibniz Institute DSMZ-German Collection of Microrganisms and Cell Cultures in Braunschweig, Germany, compared the performance of six commercial DNA extraction kits (five DNA extraction kits and one plasmid DNA extraction kit) and their suitability for purification of microbial DNA designated for MiSeq sequencing (Becker et al., Scientific Reports 6:28063).

Missed purity targets

The team ordered two kits based on anion exchange columns, two salting-out kits, one operating with magnetic silica beads and a silica spin column kit, to extract DNA from the pathogenic microorganism Klebsiella pneumoniae; known to harbour a 5,278 kb chromosome, one large plasmid (362 kb) and two small plasmids (4.8 kb and 3.8 kb). The group summarised their results in a table depicting purity, yield, cell count, completion and hands-on-time as well as cost per sample.

Astonishingly, only two kits met the A260/A280 absorbance ratio of 1.8 to 2.0, which is not only recommended for NGS-library preparation but also for many other downstream applications of extracted DNA. Three kits missed the 1.8 to 2.0 target slightly, one kit failed miserably with a A260/A280 ratio of 1.58. According to the group, however, the poor A260/A280 ratios had no negative effect on MiSeq sequencing results.

Depletion of plasmid DNA

The yields of extracted DNA varied considerably between the tested kits. Interestingly, one of the salting-out kits delivered the highest, the other one the lowest yield of all tested kits (except for the miniprep kit which is expected to provide only low amounts of DNA).

Sequencing coverage was generally higher for the two small plasmids compared to the chromosomal DNA. However, the coverage rates of both small plasmids were seven- to twelve-fold smaller with salting-out kits compared to the other kits, pointing to a depletion of plasmid DNA during the salting-out process.

Guided by other factors

But none of these slight differences in kit performance negatively influenced the sequencing results. Hence, Nübel’s team came to the conclusion: “In cases where a differential coverage of smaller plasmids (< 5 kb in our case) may be considered negligible, the choice of DNA extraction kit can be guided largely by other factors including extraction costs, extraction time and potential for automation.”

Extraction times were pretty much the same for all compared kits, ranging from 35 minutes to one hour hands-on-times and two to eight hours to complete DNA extractions from three samples. Most of the compared kits are also similarly well-suited for automation.

So, what about the last factor left then, the extraction costs? The data shown in the table of the Nübel group are pretty clear: costs per sample of the two salting-out kits were considerably lower compared to that of the silica-based spin-column kits – putting salting-out kits into the spotlight of price-sensitive researchers.

First published in Labtimes 04/2017. We give no guarantee and assume no liability for article and PDF-download.

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