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

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

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

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

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

Supramolecular assembly of the beta-catenin destruction complex and the effect of Wnt signaling on its localization, molecular size, and activity in vivo

<div><p>Wnt signaling provides a paradigm for cell-cell signals that regulate embryonic development and stem cell homeostasis and are inappropriately activated in cancers. The tumor suppressors APC and Axin form the core of the multiprotein destruction complex, which targets the Wnt-effector beta-catenin for phosphorylation, ubiquitination and destruction. Based on earlier work, we hypothesize that the destruction complex is a supramolecular entity that self-assembles by Axin and APC polymerization, and that regulating assembly and stability of the destruction complex underlie its function. We tested this hypothesis in <i>Drosophila</i> embryos, a premier model of Wnt signaling. Combining biochemistry, genetic tools to manipulate Axin and APC2 levels, advanced imaging and molecule counting, we defined destruction complex assembly, stoichiometry, and localization in vivo, and its downregulation in response to Wnt signaling. Our findings challenge and revise current models of destruction complex function. Endogenous Axin and APC2 proteins and their antagonist Dishevelled accumulate at roughly similar levels, suggesting competition for binding may be critical. By expressing Axin:GFP at near endogenous levels we found that in the absence of Wnt signals, Axin and APC2 co-assemble into large cytoplasmic complexes containing tens to hundreds of Axin proteins. Wnt signals trigger recruitment of these to the membrane, while cytoplasmic Axin levels increase, suggesting altered assembly/disassembly. Glycogen synthase kinase3 regulates destruction complex recruitment to the membrane and release of Armadillo/beta-catenin from the destruction complex. Manipulating Axin or APC2 levels had no effect on destruction complex activity when Wnt signals were absent, but, surprisingly, had opposite effects on the destruction complex when Wnt signals were present. Elevating Axin made the complex more resistant to inactivation, while elevating APC2 levels enhanced inactivation. Our data suggest both absolute levels and the ratio of these two core components affect destruction complex function, supporting models in which competition among Axin partners determines destruction complex activity.</p></div

Wg signal and GSK3/Zw3 activity are important for destruction complex membrane recruitment and GSK3/Zw3 regulates release of Arm from the destruction complex.

<p>(A,B) Localization of Axin:GFP in stage 9 sibling control embryo (A) and <i>wg</i>^<i>IG22</i> mutant (B). Neurotactin serves as a membrane marker. Both the patterned recruitment of Axin:GFP puncta to the membrane and elevation of cytoplasmic pool of Axin:GFP in Wg-ON cells (double arrows) are lost in <i>wg</i>^<i>IG22</i> mutants. (C) Stage 9 embryo ubiquitously expressing Wg, using the MatGAL4 driver driving both UAS-Wg:HA and UAS-Axin:GFP. Now all cells accumulate Axin:GFP in membrane puncta and also accumulate elevated levels of Axin:GFP in the cytoplasm. (D-F) Stage 9 <i>zw3</i> maternal/zygotic RNAi embryos expressing UAS-Axin:GFP, both driven by matGAL4 drivers (= zw3 RNAi x Axin in Methods). (D) Arm levels are highly elevated in all cells. (E,F) Membrane recruitment of Axin:GFP puncta in Wg-ON cells is lost, and Arm accumulates in Axin puncta (F, arrowheads). (G) Immunoblot with anti-Axin antibodies and quantification. Axin levels remain unchanged after <i>zw3</i> RNAi (note: UAS:Axin:GFP was not present in this cross = zw3 RNAi in Methods). * = 100 kDa band is non-specific cross-reacting band, as is indicated by the Axin RNAi control. Tubulin was a loading control. A one-way t-test was used to assess the significance of difference in Axin levels. Scale bars = 15μm.</p

The destruction complex contains thousands of APC2 or Axin molecules after over-expression in SW480 cells, and 10-100s of Axin molecules in vivo in embryos.

<p>(A) Representative images of live samples used for fluorescence comparisons to calculate GFP molecule numbers. Each panel is scaled to the same size and brightness. Ndc80:GFP assembles into a structure containing ~306 GFP molecules while Mif2:GFP assembles into a structure containing ~58 GFP molecules. (B) Pattern of Axin:GFP accumulation and localization in a live embryo. Comparison to our fixed samples allowed identification of regions receiving Wg signal (dimmer puncta) or not receiving Wg signal (brighter puncta). (C-E) Estimated number of GFP molecules per punctum. Each dot = an individual punctum analyzed. Means and standard deviation are in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007339#pgen.1007339.s016" target="_blank">S8 Table</a>. (C) GFP Molecule counts from SW480 colorectal cancer cells expressing Axin:GFP alone, Axin:GFP plus RFP:APC2, or GFP:APC2 in addition to Axin:RFP. (D-E) GFP molecule counts <i>in vivo</i> from stage 9 embryos expressing RFP and Axin:GFP under the control of MatGAL4 (Mat RFP&Axin). (E) Quantification of puncta GFP molecule counts from D, after being separated into those in presumptive regions receiving or not receiving Wg signals (as in B). Statistical analysis via an unpaired t-test.</p