Different RNase concentrations do not reduce DNA yield in Miraprepped samples and Miraprep is not significantly contaminated by low molecular weight RNA.

<p>(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).</p

Miraprepped plasmids can be effectively used to transfect human tissue culture cells.

<p>(A) Immunofluorescence of human SW480 cells transfected with a plasmid encoding GFP (3 kb; Miraprepped using 1x volume of ethanol+50 μg/ml RNase) and stained for β-catenin via antibody. SW480 cells have high levels of the Wnt transcriptional co-activator β-catenin due to a mutation in one of its key negative regulators, APC. (A’) GFP is uniformly distributed throughout transfected cells. (A”) Expression of GFP does not alter β-catenin levels—arrows compare a transfected and an untransfected cell. (B) Immunofluorescence of SW480 cells transfected with a plasmid encoding GFP-tagged <i>Drosophila</i> APC2 (8 kb; Miraprep using 1x volume of ethanol+50 μg/ml RNase). (B’) APC2 is uniformly distributed in the cytoplasm. (B”) Fly APC2 is able to reduce β-catenin levels, thus compensating for the mutation of the endogenous human APC in the SW480 cells. (C) Transfection efficiency into SW480 cells is similar for Miraprepped samples (using 1x volume of ethanol+50 μg/ml RNase) and those transfected with DNA prepared via the standard Qiagen Maxiprep procedure. 2 μg of plasmids encoding GFP (3 kb) or GFP-tagged Drosophila APC2 (8 kb) were transfected using Lipofectamine 2000. 100 cells were counted in each of three independent experiments. (D) Immunoblot analysis of transfection efficiency. 3 conditions were tested: DNAs prepared by Miniprep (GeneJET), Miraprep (using 1x volume ethanol), and Miraprep (using 1x vol+50 μg/ml RNase). All led to roughly equal levels of protein expression. Lipofectamine 2000 and 2 μg of plasmid DNA were used. Cells were directly lysed in SDS-loading buffer. aPKCγ was used as the loading control.</p

The Miraprep: A Protocol that Uses a Miniprep Kit and Provides Maxiprep Yields

<div><p>Plasmid purification is a basic tool of molecular biologists. Although the development of plasmid isolation kits utilizing silica spin columns reduced the time and labor spent on plasmid purification, achieving large plasmid DNA yields still requires significant time and effort. Here we introduce the Miraprep, a rapid protocol that allows isolation of plasmid DNA using commercial Miniprep kits, but with DNA yields comparable to commercial Maxiprep plasmid purifications. Combining ethanol precipitation with spin column purification, we created a DNA isolation protocol that yields highly concentrated plasmid DNA samples in less than 30 minutes. We show that Miraprep isolated plasmids are as stable as plasmids isolated by standard procedures, can be used for standard molecular biology procedures including DNA sequencing, and can be efficiently transfected into mammalian cells. This new plasmid DNA isolation protocol will significantly reduce time and labor without increasing costs.</p></div

Comparison of commercial plasmid preparation methods from three manufacturer’s (GeneJet, Qiagen, and GenElute) with the Miracle-prep.

<p>Comparison of commercial plasmid preparation methods from three manufacturer’s (GeneJet, Qiagen, and GenElute) with the Miracle-prep.</p

Testing whether the columns might act as a filter and verifying DNA yields using comparison to known DNA standards.

<p>(A) To determine whether silica spin columns might capture DNA by acting as filters, the Miraprep procedure was followed through the neutralization step with 1x volume of ethanol added or no ethanol added as a control, and then the sample was passed over a simple centrifugal filter (pore size 0.22 μm), the filter was washed following the Miraprep protocol, DNA was eluted from the top surface of the filter, and electrophoresed on an agarose gel. DNA was only recovered after ethanol addition. (B) Silica columns are more efficient than centrifugal filters in capturing plasmid DNA. Comparison of DNA yields using silica columns or centrifugal filter columns; from three independently conducted Mirapreps. (C,D) Standard Miniprep plasmids, or Miraprep plasmids prepared using our final protocol using 1x volume of ethanol, were electrophoresed on an agarose gel and amounts compared to known DNA standards (Thermo Scientific GeneRuler 1 kb DNA Ladder #SM0312 (0.5 μg/μl)). 2 μl DNA plus 5 μl loading buffer were loaded in each lane. (C) GenElute kit. (D) GeneJET kit. Above each gel is the DNA amount calculated from OD260 and below the gel estimates from comparison to DNA markers of known amounts. Image J was used to quantify DNA band intensities in (C,D). DNA amounts calculated by both methods were comparable.</p

Miraprepped samples are stable and can be used for sequencing.

<p>(A) Plasmids prepared using the Miraprep protocol with 1x volume of ethanol+50 μg/ml RNase are as stable as commercial Miniprepped plasmids after incubation overnight at 37°C. 2 μl of Mira- or Miniprepped samples were loaded. (B) Sequencing reaction of a Miniprepped APC2 (8 kb) plasmid—the sequence from 683 base pairs (bp)—712 bp is shown. 0.7 μg of DNA was used for the sequencing reaction (C) Sequencing of Miraprepped APC2 plasmid. 0.7 μg of DNA was used, and the same region as in (B) is shown.</p

Addition of Ethanol leads to increased plasmid DNA yield.

<p>(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.</p

Neither Roc1b nor Roc2 is individually required for regulating Arm levels in embryos or larvae.

<p>Antigens and genotypes indicated. A–G. Embryos, anterior left. A. In wild-type stage 9–10 embryos segmentally repeated groups of cells receive Wingless signal, stabilizing Arm in the cytoplasm and nuclei (arrow). In other cells, Arm outside adherens junctions is destroyed (arrowhead). B. In <i>APC2 APC1</i> maternal/zygotic double mutant embryos, Arm levels are highly elevated, exceeding those in any cells in a wild-type embryo. C. When one induces clones of <i>APC2 APC1</i> double mutant cells in the developing larval brain (double mutant cells are marked with GFP using the MARCM technique), cytoplasmic Arm levels are modestly elevated (arrows) relative to wild-type cells (arrowhead). D–G. Stage 9 wild-type (D,F), <i>Roc1b^dc3</i> maternal/zygotic (E), or <i>Roc2^KG</i> maternal/zygotic embryos (G). For each mutant, wild-type embryos marked with Histone-GFP were stained in the same tube as mutants. Arm accumulation remains unchanged in both mutants. H–K. 3^rd instar wing imaginal discs from wild-type (H,J), <i>Roc1b^dc3</i> zygotic mutants (I), or <i>Roc2^KG</i> zygotic mutants (K). L–O. 3^rd instar larval brains from wild-type (L,N), <i>Roc1b^dc3</i> zygotic mutants (M), or <i>Roc2^KG</i> zygotic mutants (O). In both wing discs and brains no changes in Arm accumulation were apparent in either mutant. P. Immunoblot of cell extracts made from stage 9 embryos. Tubulin serves as a loading control. Scale bar = 50 µm.</p

<i>APC2³³</i> has a hypomorphic phenotype.

<p>A–F. 3^rd instar wing imaginal discs. G–K. 3^rd instar larval brains. Clones of mutant cells of the indicated genotype were induced using the MARCM method <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031284#pone.0031284-Lee1" target="_blank">[42]</a> and homozygous mutant cells are marked by the presence of GFP. A–B. Cells in the wing pouch that are <i>APC2^g10 APC1^Q8</i> double mutant accumulate modestly elevated levels of Arm (arrows), while mutant cells in regions surrounding the wing pouch segregate and form cysts (arrowheads). C,D. In contrast, cells in the wing pouch that are <i>APC2³³ APC1^Q8</i> double mutant do not accumulate elevated levels of Arm (arrows), and mutant cells in regions surrounding the wing pouch do not always segregate to form cysts (arrowheads). E,F. Clones of cells that are <i>APC2^19-3 APC1^Q8</i> double mutant (E) or <i>APC2³³ APC1^Q8 Axin</i> triple mutant (F) behave like <i>APC2^g10 APC1^Q8</i> double mutant cells. G. Neurepithelial cells in anterior medullar region of the larval brain that are <i>APC2^g10 APC1^Q8</i> double mutant accumulate modestly elevated levels of Arm (arrow) and segregate from neighbors, in contrast to neighboring wild-type cells (arrowhead; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031284#pone.0031284-Hayden1" target="_blank">[18]</a>). H. Neurepithelial cells in anterior medullar region of the larval brain that are <i>APC2^19-3 APC1^Q8</i> double mutant behave similarly to <i>APC2^g10 APC1^Q8</i>. I. Neurepithelial cells in anterior medullar region of the larval brain that are <i>APC2³³ APC1^Q8</i> double mutant sometimes segregate but do not always accumulate elevated Arm levels (arrow vs. arrowhead). J. Medullar neurons that are <i>APC2^g10 APC1^Q8</i> double mutant invariably send out axons into the center of the clone, forming axonal knots (arrow; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031284#pone.0031284-Hayden1" target="_blank">[18]</a>) instead of the normal finely fasciculated projections (arrowhead) to the medullar neuropil <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031284#pone.0031284-Hayden1" target="_blank">[18]</a>. K. Some medullar neurons that are <i>APC2³³ APC1^Q8</i> double mutant do not form axonal knots but instead send normal projections to the medullar neuropil (arrows). Scale bars = 50 µm.</p

A canonical SCF complex including Roc1a regulates Arm levels in <i>Drosophila</i> S2 cells.

<p>Immunoblots of cell extracts from S2 cells treated with double-stranded RNA targeting the genes indicated. Tubulin and the septin Peanut serve as loading controls. The “SK” negative control is double-stranded RNA directed against the bacterial plasmid pBluescriptSK. A. Of the Cullins, only Cullin1 RNAi elevates Arm levels. B. Antibodies were available to confirm knockdown of Cullin1, Cullin4 and Cullin 5. All were significantly knocked down. These control samples were also used in the parallel screen for SCF proteins that regulate centrosome number, which was published in the Journal of Cell Biology <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031284#pone.0031284-Rogers1" target="_blank">[23]</a>. C. RNAi directed against both SkpA and SkpB elevates Arm levels—to confirm the role of SkpA, we used RNAi directed against the non-conserved 3′ UTR. D. SkpB RNAi also reduces SkpA levels, presumably due to sequence similarity. E. Roc1a RNAi elevates Arm levels, as does triple RNAi against all three Rocs. SK RNAi serves as a negative control and RNAi against Zw3 (fly GSK3) as a positive control. F. Diagram of primers used to generate different dsRNAs against Roc1, some of which are non-overlapping, to test for off-target effects. G. RNAi against the 5′ or 3′ half of the Roc1a mRNA each lead to similar elevation of Arm levels as is caused by RNAi against the entire coding sequence. H,I. RNAi against each of the F-box proteins that scored positive in the primary screen, plus Ebi (which previously was reported to have a role in ßcat stability), Ago, and Ppa. Only Slimb RNAi elevated Arm levels.</p