Adding ethanol after the neutralization step increases yields from commercial silica spin column-based DNA Miniprep kits

Commercial Maxiprep kits require a large time investment. This led us to explore alternate methodologies to reduce the preparation time, but still maintain high yields of DNA. Ethanol can act to dehydrate DNA, which is predicted to increase interaction with silica, and for this reason a number of commercial kits include alcohols in the binding or wash buffers. We thus explored whether adding ethanol after the alkaline lysis and neutralization steps and before loading on the column might increase yields of commercial Miniprep kits. We first explored how varying the ethanol concentration affected both yield and DNA purity. We examined three different size high copy plasmids: a 3 kb plasmid, pmaxGFP, which encodes standard GFP, and 8 kb and 14 kb plasmids sharing the same backbone, pEGFP-N1, and encoding Drosophila Adenomatous polyposis coli2 (APC2) or human APC, respectively [9] (the plasmid suppliers estimate backbone copy number of 500/cell). We initially tried two different commercial Miniprep kits: GeneJet and QIAGEN. DNA yield of all three size plasmids increased significantly when 1x volume of ethanol was used, while 1.5x or 2x volumes of ethanol provided the highest apparent DNA yield, as assessed by the OD260 (Fig 1A and 1B, top). We next tested DNA purity by calculating the OD260/280 ratio (Fig 1A and 1B, bottom). Pure DNA has a ratio of 1.8, and thus a ratio of 1.75–1.95 is generally considered to be a good DNA preparation [10]. A ratio >1.95 indicates RNA contamination while a ratio <1.7 indicates protein contamination. The 1x volume Miraprep samples had OD260/OD280 ratios of 1.81–1.91, while samples with 1.5x volumes of ethanol were >1.91, suggesting RNA or other types of contamination in the latter. When DNA yields were assessed by gel electrophoresis and comparison of the plasmid bands to known DNA standards, 1x volume Miraprep samples had DNA concentrations consistent with their OD260, similarly to what we saw with Miniprep or Maxiprep samples (see below), while 1.5x volume Miraprep samples were inconsistent relative to their DNA amount determined by gel electrophoresis (data not shown). Thus a 1x volume of ethanol for DNA precipitation was selected. We further verified that this protocol using 1x volume of ethanol increased DNA yields when using a third commercial spin column Miniprep kit, Sigma GenElute (Fig 1C).

PPT PowerPoint slide

PowerPoint slide PNG larger image

larger image TIFF original image Download: Fig 1. Addition of Ethanol leads to increased plasmid DNA yield. (A) DNA plasmid preps of the indicated plasmids with different concentrations of ethanol. Top: DNA concentration as assessed by OD260, middle: 2 μl of each sample was electrophoresed on an agarose gel and visualized by ethidium bromide staining, bottom: OD260/280 ratio. The GeneJET Plasmid Miniprep kit was used. (B) As in (A) but the Qiagen Miniprep kit was used. (C) Plasmid preparations with the GenElute kit, comparing either the standard Miniprep procedure or the Miraprep (using 1x volume of ethanol). The Mirapreps in (C) included fresh addition of RNase (50 μg/ml) as in the final Miraprep protocol, and values are the average of two experiments, showing mean and standard deviation. https://doi.org/10.1371/journal.pone.0160509.g001

The differences in OD260/OD280 ratios seen with different amounts of ethanol addition suggested the possibility of RNA contamination. Consistent with this, when we compared older Miniprep kits, in which RNase was added to the resuspension buffer several weeks earlier, with new Miniprep kits, where the manufacturer’s RNase was freshly added, DNA yields as assessed by OD260 sometimes were higher than those determined by comparison to DNA markers of known concentrations analyzed via agarose gel electrophoresis (data not shown). This suggested that the RNase activity in the resuspension buffer might become depleted over time and become insufficient to decrease RNA levels efficiently. We thus explored whether fresh RNase addition might alleviate this issue. Fresh RNase addition into the Miraprep resuspension buffer at 0–100 μg/ml did not reduce DNA yields, as assessed by gel electrophoresis (Fig 2). Thus to achieve consistent and effective RNA depletion in Miraprep samples the final protocol includes adding fresh 50 μg/ml RNase to the resuspension buffer before each preparation. We verified that our modified Miraprep eliminated the low-molecular weight RNAs present in the initial preparation as well as they were removed by a standard Miniprep. To do so, we examined by gel electrophoresis the initial cell lysate after alkaline lysis and the spin to remove cellular debris and high molecular weight DNA (Fig 2B and 2C Pre-column), the initial flow-through (Fig 2B and 2C Flow-through), and the final eluted plasmid DNA (Miniprep or Miraprep). While low-molecular weight RNAs were clearly present in the initial lysate, they were at very low to undetectable levels in the eluted plasmid DNA, suggesting that our estimated Miraprep plasmid yields are not significantly altered by contaminating small molecular weight RNAs (this is also consistent with the efforts below to estimate plasmid yield directly by gel electrophoresis and comparison to known DNA standards).

PPT PowerPoint slide

PowerPoint slide PNG larger image

larger image TIFF original image Download: Fig 2. Different RNase concentrations do not reduce DNA yield in Miraprepped samples and Miraprep is not significantly contaminated by low molecular weight RNA. (A) Standard Miniprep, or Miraprepped plasmids prepared using 1x volume of ethanol, were treated with indicated RNase concentration, added freshly into the resuspension buffer before beginning the procedure. Top: 0.4 μg was electrophoresed on an agarose gel. DNA concentration only varied slightly when RNase was freshly added. Bottom: OD260/280 ratio. (B,C) Testing for low molecular weight RNA in Miniprep and Miraprep samples, respectively. (B) Miraprep and Miniprep samples of the 8 kb plasmid contain little or no small molecular weight RNA. Pre-column = after alkaline lysis, Flow-through = flow-through of spin column, Final lane in each set is eluted plasmid. 10 μl of pre-column and flow-through samples were loaded, while 2 μl were loaded of Miniprep or Miraprep samples. (C) Miniprep and Miraprep samples of the 14 kb plasmid have little to no low molecular RNA present. Loading same as described in (B). https://doi.org/10.1371/journal.pone.0160509.g002

One possible mechanism by which the Miraprep procedure could increase DNA yields was that the ethanol addition led to DNA precipitation and that the silica column acted, at least in part, as a filter to capture the precipitated DNA. To determine whether this was plausible, we went through our procedure to the neutralization step, added 1x volume of ethanol or no ethanol as a control, and then ran the sample over a simple centrifugal filter (pore size 0.22 μm). We then eluted DNA from the top surface of the filter. Consistent with the idea that the column acts as a filter, we were able to effectively capture DNA on the centrifugal filter only from samples where ethanol was added (Fig 3A). However, DNA recovery from the centrifugal filter was not as effective as that from the silica columns (Fig 3B), suggesting that the columns may act as more than just a filter.