text:
- Method
- Open Access
POSaM: a fast, flexible, open-source, inkjet oligonucleotide synthesizer and microarrayer
© Lausted et al.; licensee BioMed Central Ltd. 2004
- Received: 21 April 2004
- Accepted: 24 June 2004
- Published: 27 July 2004
DNA arrays are valuable tools in molecular biology laboratories. Their rapid acceptance was aided by the release of plans for a pin-spotting microarrayer by researchers at Stanford. Inkjet microarraying is a flexible, complementary technique that allows the synthesis of arrays of any oligonucleotide sequences de novo. We describe here an open-source inkjet arrayer capable of rapidly producing sets of unique 9,800-feature arrays.
- Additional Data File
- Propylene Carbonate
- Phosphoramidite
- Oligonucleotide Synthesis
- Print Head
The DNA array is a powerful tool for the high-throughput identification and quantification of nucleic acids. Among other uses, array analysis has become a standard technique in the molecular biology laboratory for monitoring gene expression. Arrays can be made either by the mechanical spotting of presynthesized DNA products [1] or by the de novo synthesis of oligonucleotides on a solid substrate, usually a derivatized glass slide. As the sequences for de novo synthesized arrays are stored electronically rather than physically in frozen DNA libraries, the costs and the potential for errors in amplification, storage, and retrieval are eliminated.
Piezoelectric inkjet oligoarray synthesis as proposed by Blanchard and Hood [2] is a particularly flexible method that allows the rapid construction of oligonucleotide arrays containing any desired sequence. By dispensing DNA monomers from a multi-channel inkjet print head, large numbers of sequences can be chemically synthesized in parallel. Development of a prototype inkjet system began in our laboratory and was continued in the private sector [3, 4]. Work by Rosetta Inpharmatics (Seattle, WA) and Agilent (Palo Alto, CA) resulted in an industrial-scale inkjet oligoarraying system and a catalog of commercially available arrays. However, there has been virtually no access to the inkjet synthesis instrumentation in basic, academic research laboratories, and high set-up fees still deter the production of specialized arrays in small batches.
When compared to the rapid acceptance of the pin-spotting microarrays [5–8], the lack of access to oligonucleotide microarray synthesizers has hampered the development of new uses for this technology. The early release of the Stanford pin-spotting arrayer design [1] also spurred commercial development of high-quality microarrayers, which led to the availability of numerous models at competitive prices. Despite the problems and errors associated with collection of DNA libraries, the widespread availability of pin-spotted microarrays led to acceptance and standardization of protocols, terminology, data storage, and analysis techniques [9–11].
Several laboratories have modified commercial ink-on-paper printers for use in spotting microarrays. These printers were usually based on 'bubble jet' print heads containing tiny heating elements that rapidly vaporize a water-based solution in a capillary to eject a droplet containing protein or DNA, onto a solid support [12–14]. These printers, however, are difficult to clean and are not suitable for high-throughput production of oligoarrays or the parallel de novo synthesis of oligonucleotide arrays. Like pin-spotting microarrayers, they also require libraries of known nucleic acid reporters before arrays can be made.
The first successful technique for de novo oligonucleotide synthesis on a chip was developed by Affymetrix (Santa Clara, CA) using photolithographic techniques borrowed from the semiconductor industry. By using photomasks and UV-catalyzed base deprotection, large numbers of oligonucleotide arrays can be synthesized with a high feature density. Chips are now being made that contain all the genes predicted to be in the human genome. However, coupling chemistry using photolabile monomers is less efficient than standard phosphoramidite chemistries, resulting in arrays consisting of short oligonucleotides (25 mers) that require multiple features for the unambiguous identification of each gene. In addition, the design and construction of new arrays is slow and expensive because new masks need to be cut for each base changed in the array.
Recently, new photolithographic oligoarray synthesizers have been introduced that replace mask sets with a dynamic micro-mirror device (DMD) [15, 16]. NimbleGen (Madison, WI) produces arrays with up to 390,00 features using Affymetrix-style chemistry. These arrays are not currently available for sale in the United States, but samples can be sent to Iceland for hybridization and scanning. Instead of deprotecting photolabile monomers, Xeotron (Houston, TX) uses the DMD to photo-generate detritylating acids. This has the advantage of better yields through higher coupling efficiency, but feature density is lower. Xeotron arrays currently contain about 8,000 features. Detailed descriptions of the DMD-based instruments are not publicly available.
De novo oligonucleotide synthesis using a piezoelectric inkjet oligoarray synthesizer overcomes several of the problems inherent in the pin-spotted arrays and the conventional photolithographic mask arrays. First, as soon as the genomic DNA or expressed sequence tag (EST) library is even partially sequenced, oligonucleotide reporters, including intergenic regions, can be designed and synthesized on arrays without having to clone and store large libraries. Second, the use of standard phosphoramidite chemistry for oligoarray synthesis allows longer reporters to be synthesized, decreasing the number of reporters required for confident identification of the target molecules. The POSaM (piezoelectric oligonucleotide synthesizer and microarrayer) platform described here utilizes a low-cost piezoelectric print head with six fluid channels; four channels deliver phosphoramidite precursors and one delivers an activator (ethylthiotetrazole), leaving one channel available for an optional linker or modified base. The piezoelectric jets can deliver a wide range of nonvolatile solvents in volumes as low as 6 pl. Piezoelectric jetting, high-quality motion controllers and standard phosphoramidite oligonucleotide synthesis chemistry allow users to synthesize arrays of any nucleic-acid sequence at specific, closely spaced features on suitable solid substrates.
We present here the description of a simple oligoarray synthesis platform along with the plans for its construction and a detailed user manual. The POSaM platform can produce multiple unique microarrays, each with 9,800 different reporter sequences, on modified glass microscope slides. Synthesis is inexpensive and rapid; new arrays can be ready for hybridization the same day that they are designed. These arrays are suitable for a wide range of biological investigations including the study of gene expression, alternative splicing, chromatin immunoprecipitation techniques (ChIP-to-Chip), and single-nucleotide polymorphism (SNP) detection. In addition, the covalent linkage of the oligonucleotides to the substrate is sufficiently robust to allow for re-use of individual oligoarrays without a detectable change in the signal-to-noise ratio.
Description of the instrument
Cost of materials required to make one inkjet oligonucleotide array
Price ($)
Line cost ($)
Glass slides
Nano-Strip glass cleaner
Sodium hydroxide
Hydrochloric acid
Rain-X silane solution
Slide derivatization subtotal
0.02 M Iodine THF/Pyr/H2O
2.5% DCA in DCM
dA-CE phosphoramidite
Ac-dC-CE phosphoramidite
dG-CE phosphoramidite
dT-CE phosphoramidite
2-methyl glutaronitrile
Chemical synthesis subtotal
Lifterslip coverslips
DIG Easy-Hyb solution
Control oligo (Bodipy)
Wash buffers
Test hybridization subtotal
Syringe, 1 ml
Syringe, 5 ml
26G needles
Molecular sieves
Inkjet head
Exhaust filter cartridge
Miscellaneous materials subtotal
Motion control and printing
The three-axis positioning system utilizes Parker-Daedal 506-series ball screw linear actuators driven by servo motors (Olympic Controls, Wilsonville, OR) that can move the print head over a wide range with 5 μm repeatability. The Epson F057020 six-color print head has six banks of 32 nozzles, or 192 individually addressable piezoelectric pumps. We purchased one complete Photo 700 printer (Epson America, Long Beach, CA) to obtain a print-head holder and ribbon cable, and subsequent print heads from Agson Electronics (Cherry Hill, NJ).
Droplet detection
Reagent/solvent storage and delivery
Reagents and waste are stored in glass bottles with GL-45 screw-top caps. Amber 500 ml bottles hold oxidizer and deprotection acid, while clear 2 l bottles hold acetonitrile and waste. Pressurizing nitrogen enters the reagent and solvent bottles through PTFE check valves and the waste bottle is under vacuum. Six PTFE solenoid valves (Model 190224S30, Angar, Florham Park, NJ) are used to control the flow of acetonitrile, oxidizer, acid, waste and other reagents.
Solvents for piezoelectric oligonucleotide synthesis
Inert gas supply and system enclosure
Inert gas and pressurization is supplied to the system by a liquid nitrogen dewar. The whole instrument is enclosed with a custom acrylic cover with an internal volume of approximately 510 l. The cover is transparent and airtight. It has a single latched access door and a glove panel that allows operators to work inside the enclosure without opening the access door. The inert gas flow is controlled by a 0-100 liters per minute valve/rotameter. This gas supply displaces the air inside the enclosure and powers the air amplifier that circulates the internal atmosphere through a series of activated charcoal and desiccant filters. The levels of moisture and oxygen, both toxic to phosphoramidite synthesis, must be minimized. Nitrogen is flushed continuously through the enclosure starting at least 40 minutes before synthesis and ending only after the last cycle. Humidity is logged automatically by the machine; oxygen levels are checked periodically by the operator. Levels are maintained below the instruments' limits of detection.
Slide holder
The slide holder (Figure 1b) was machined from a solid block of PTFE with O-ring inserts so that 27 slides can be secured in place by a vacuum. Inclined troughs beneath the slides aid in collection and removal of waste reagents. New designs for the slide holder that improve printing and washing speed are currently being investigated.
System performance
The Lombardi software was designed to fire each piezoelectric pump on the fly as the print head passes over the slides. Before each round of printing the software examines the operation of each of the 192 pumps (see QC description above) and determines the optimal movement of the print head and firing order of the piezoelectric pumps. The time for array synthesis depends primarily on the length of the sequences being printed, and to a lesser degree on the number of slides being printed, the number of features on the chip, and the number of nozzle failures. Currently, the rate-limiting steps in oligoarray synthesis are the washing and drying of the slides. Nozzle failures can be minimized by flushing the print head with pure solvent immediately after completion of array synthesis. Nozzle failures have not been a significant problem using the 1:1 MGN:3MP solvent system. The median number of nozzle failures is 3 out of 192. As the print heads are very cheap, it is our practice to replace them every three months. This is more of a precautionary step rather than being required by an accumulation of nozzle failures; at three months it is unusual to have more than a few plugged nozzles.
Reporter spot diameter
The average diameter for each feature was determined by measuring the dimensions of 40 oligonucleotide features chosen at random from four arrays printed on Xenopore (Hawthorne, NJ) Xenoslide E epoxy slides. After printing and hybridization with a complementary, fluorescence-labeled oligonucleotide, the arrays were scanned with a confocal laser scanner and the diameters were determined using ImageJ software [17]. The slides produced a feature diameter of 156 ± 16 μm. Similar results were observed using Bioslide (Walnut, CA) Precision CT Epoxy slides. Most of the following data was obtained using Xenoslide E substrates; however, the primary source of array failure is due to inconsistencies in the slide surfaces, even within commercial slide lots. We have found that the most consistent slides, by far, are those produced by our liquid deposition protocol (described in Materials and methods). In addition, these slides are much cheaper than the commercial alternatives. This protocol was used in the yeast barcode experiment described below and is now part of our standard operating procedure.
Microarray performance
Virtual reaction wells
Direct labeling of reporters
Hybridization specificity
Partial list of sequences arrayed in hybridization experiments
T20g' perfect match
T20r' perfect match
T20r' - 1 mismatch
T20r' - 2 mismatch
T20r' - 3 mismatch
T20r' - 4 mismatch
T20r' - 5 mismatch
T12r' perfect match
T12r' 1 mismatch
T12r' 2 mismatch
T12r' 3 mismatch
T12r' 4 mismatch
YPL110C match
YPL110C mismatch
YPL111W match
YPL111W mismatch
YPL112C match
YPL112C mismatch
Array re-use
Hybridization with deletion-strain barcodes
With the specificity and re-usability of inkjet arrays validated by hybridization with oligonucleotide targets, we tested arrays with a complex sample of PCR products prepared from the genomic DNA of 94 yeast gene-deletion strains. Accurate oligoarray measurement of deletion barcodes will greatly simplify the analysis of growth phenotypes. For example, a competitive growth condition may be applied to a pooled culture containing equal proportions of each strain. The oligoarray can measure changes in the quantity of each barcode strain and, therefore, the change in each population from initial to final culture [22, 23].
A major drawback of conventional microarray fabrication is the time and cost required to develop a new array. When a new genome is sequenced, when additional expressed genes are identified, or when polymorphisms are discovered, a great deal of time and money is required to create a new microarray. The photolithographic technique, such as that used by Affymetrix [24], requires the fabrication of up to 100 chrome/glass masks for de novo synthesis of a new 25 mer oligonucleotide array. Spotted arrays can only be fabricated after the characterization of thousands of DNA fragments is completed, and the reporters have to be replicated, cataloged, and stored. New POSaM arrays, however, can be designed, synthesized, hybridized and scanned in a single day.
Key properties of the POSaM platform is that fabrication of a new array is flexible and rapid, and cost is practically independent of the number of sequences or the number of different arrays to be synthesized. Once the inkjet is running, it does not matter if the arrays contain 100 reporters or the current maximum of 9,800 features; there is no additional set-up cost for a new design other than the time it takes for the reporter selection process. The cycle times for covalent attachment of each base in a reporter ranges from 11 minutes for one array to 20 minutes for eight arrays. Six arrays of 40 mers can be produced in about 13 hours. As each oligonucleotide reporter can be of a different length, it is possible, by varying the length of the reporter, to design arrays where all the theoretical melting temperatures are very similar. Inkjet oligoarray synthesis uses highly efficient, well characterized phosphoramidite chemistry with sequences that are stored in a database rather than in a freezer. This eliminates the cost of amplification, replication, storage and retrieval of the reporters, as well as minimizing the errors that are generated when cataloging many large DNA libraries.
Inkjet arrays are particularly well suited for applications where small numbers of arrays are required or where rapid reporter redesign is likely. A prime example is validation of reporters used for gene-expression arrays. Despite continual efforts to determine the definitive computational algorithms for designing unique oligonucleotide sequences that will specifically identify and quantify the amount of mRNA present in a given sample, many of the reporters predicted by these programs do not work. Ultimately, final reporter selection usually requires investigators to determine empirically which reporters work with the different samples used. It is desirable to know that a reporter set has sufficient specificity and sensitivity to detect genes of interest before many weeks of time and thousands of dollars are invested in large-scale synthesis and purification of the DNAs or fabrication of the masks needed for photolithographic synthesis. As the turnaround time for new arrays is so fast and the cost of printing 9,800 reporters is so small with the POSaM platform, many different oligonucleotide sequences can be tested to optimize the sensitivity and specificity of the reporters. We estimate that it costs (including labor) approximately $300 to synthesize a set of six arrays (the number of arrays that is easily produced in a single day). Material costs are itemized in Table 1. These arrays could be identical or contain completely different reporters, as the number of different sequences printed does not change the cost or speed of synthesis.
The fact that the arrays may be stripped and re-used will be very helpful for optimizing hybridization conditions. Specificity and sensitivity can be measured under different buffer and temperature conditions on the same array to reduce cost. Because the same sample would be applied in every condition, residual signal of less than a few percent would not be a problem. In theory, other types of arrays that use covalent linkers should also be re-usable. It has recently been reported that some UV-crosslinked cDNA arrays can also be reused [25].
The discovery and development of chip-based oligoarray applications other than the regulation of gene expression can benefit from the flexibility inherent in POSaM oligoarray synthesis. We show here that POSaM-based arrays can be useful for identifying changes in the population fraction of barcoded yeast deletion mutants placed under selective growth conditions. In designing the chips for this experiment, we found that the thermodynamic stability and the intensity of the signal of the duplexes was enhanced by including 15 bases from the regions flanking the 20-base barcode, but that only a little specificity was lost by including these complementary regions, although this could account for the low background of binding seen in the mismatched barcodes. For most purposes, reporters of length 30 to 35 nucleotides provides an optimum combination of sensitivity and specificity [26].
In addition, analysis of protein-binding sites in native chromatin, using ChIP-to-Chip methods [27] or DamID [28] assays require reporter sequences that include the noncoding regions of genomic DNA. Few commercial microarrays are available containing these intergenic regions because the cost of synthesizing or cloning every noncoding region in a genome is exorbitant. Inkjet arrays have been designed and printed with the POSaM platform where reporters tile across putative regulatory regions from -1,000 to +200, relative to the site of transcription initiation, with a step size as small as one base. Because the immunoprecipitated chromatin can be chemically or physically sheared into smaller fragments, the sequences complementary to the binding sites can be narrowed down to a much smaller region using arrays made on the POSaM platform (data not shown). This experimental data can supplement computational techniques to determine novel cis-acting binding sites and is currently being tested using the well defined yeast galactose utilization pathway, a model system used in our laboratory.
We are also expanding upon the methods of Bulyk et al. [29] by using the POSaM platform to synthesize 40-50-base double-stranded (ds) DNA oligonucleotides that contain a common primer sequence as well as potential cis-acting protein-binding motifs. The standard single-stranded (ss) DNA arrays can be converted into dsDNA arrays using primer extension reactions, and these unique dsDNA chips, which tile across putative regulatory regions, can be used to detect protein binding from nuclear extracts. With the availability of specific antibodies against most known transcription factors and with advances in surface plasmon resonance (SPR), atomic force microscopy (AFM), and MALDI-TOF tandem mass spectrometry (MS/MS) techniques, it should be possible to identify the proteins and protein complexes bound to the dsDNA arrays.
In a separate project, we designed and constructed a temperature-ramping microarray scanner that, when used with POSaM produced slides, can monitor the dissociation kinetics of each reporter/target on the array, achieving single-base mismatch resolution. Besides the obvious use for SNP detection, the ability to synthesize any sequence of DNA and determine the apparent dissociation constant make it possible to test the algorithms and heuristics used to select reporters, thereby increasing our understanding of how nucleic acids interact.
There are several areas where the design can be improved. First, since the QC testing of the print head with the laser droplet detector cannot take place during actual printing, intermittent nozzle failures may go undetected. Our current solution is to double-print each slide under the assumption that random nozzle failures are less likely to occur and go undetected at the same nozzle twice in a row. A more elegant solution, which we are in the process of adding, is to incorporate a real-time visual monitoring system that detects the presence or absence of droplets as well as whether the droplets are the correct shape and in the correct position. The total cost for the assembly of the POSaM platform is currently about $34,000. The real-time image analysis system that we are testing increases the cost by about $4,000. Second, the Epson Photo 700 printer has been discontinued. While new replacement print heads are still available, it is inevitable that they will become scarcer and more expensive. Fortunately, the electrical interface changes very little with each new model. We are redesigning the print head assembly to make it easier to adapt the POSaM to future print-head models. Third, the washing and drying steps are array synthesis bottlenecks. New slide-holder designs, along with modifications to the software, are being considered. Fourth, with improvements to the slide surface chemistry, it may be possible to greatly increase feature density. These are projects that we are currently investigating and that can benefit from the open-source process. We are also sure that new POSaM users will suggest additional improvements.
While de novo synthesized oligonucleotide arrays are starting to become available commercially, there remains a need for an open-source version of the inkjet oligonucleotide arrayer in the spirit of the pin-spotting instrument and software first released by the Brown and DeRisi laboratories in 1998. This open-source instrument accelerated the growth of the array field by allowing early adoption of the technology by laboratories with the desire and technical ability to construct their own instrument, provided a standard design that both laboratories and public-sector manufacturers could use to improve the instruments and methods, and it also allowed end users to take control of microarray design and decide what arrays would be available. A shared, non-proprietary arrayer allows end users to tinker freely with the design and make improvements as needed. Thus, we believe that making the POSaM platform freely available to academic laboratories will accelerate the development of better instruments. In addition, the selection of reporter sequences could be standardized, oligoarray chip applications expanded, and science would be accelerated in a positive, productive manner.
We believe that POSaM-like inkjet systems can be constructed by most well equipped molecular biology laboratories with modest organic chemistry and engineering expertise. The most unfamiliar step in the construction of the POSaM platform was the construction of circuit boards; however, once the boards in the present platform were designed, they were soldered and tested by undergraduates in our laboratory. While the surface chemistry of the substrate is still somewhat problematic, the development of the new modification protocol described in the Materials and methods brings the success rate to an acceptable 80-90% and the release of this instrument into the public domain should facilitate the development of the instrument, protocols and new uses for oligonucleotide arrays.
Materials and methods
Substrate preparation
Substrates were prepared by derivatizing clean microscope slides with 3-glycidoxypropyltrimethoxysilane (GPTMS). GPTMS was obtained from Acros Organics USA (Morris Plains, NJ). Fisher Scientific (Pittsburgh, PA) 'Premium' 25 × 76 mm microscope slides were cleaned by immersion in Nano-Strip cleaner (Cyantek, Freemont, CA) at room temperature for 30 min, followed by five rinses of 5 min each in de-ionized water. The slides were incubated overnight in 10% NaOH at room temperature and were again rinsed with de-ionized water (5 × 5 min). The slides were soaked in 1% HCl solution for 1 min at room temperature followed by rinsing in de-ionized water (2 × 5 min). The slides were oven-dried at 150°C for 90 min and cooled to room temperature under a stream of nitrogen. For liquid deposition, slides were immersed in a solution of 2.5% GPTMS in Rain-X (SOPUS Products, Houston, TX) and sonicated for 2 min. Following sonication, slides were cleaned by immersion in 100% ethanol for 2 min, de-ionized water for 5 min, and dehydrated in 100% acetone for 5 min. On removal from acetone, the slides were immediately spun dry at 1,000g and stored in a desiccating cabinet until use.
Just before arraying, the silanized substrates were washed in 100% methanol (10 min, room temperature), rinsed in de-ionized water, and rapidly dried under a stream of nitrogen. The printing area, approximately 25 mm × 25 mm in the center of the slide, was marked on the underside with a pen and the margins of the slides are treated with Rain-X to increase the hydrophobicity of the bounding regions. The highly hydrophobic margins serve to constrain the bulk washes to the active area of the slide, reducing reagent use, and aiding reagent removal.
Synthesizer operation
The inkjet oligonucleotide synthesis process uses standard phosphoramidite chemistry as described by [30], and modified for use in automated oligonucleotide synthesizers by [31]. Nucleoside phosphoramidites (bzdAMP, AcdCMP, ibudGMP, dTMP), 5-ethylthio-1H-tetrazole, and oxidizer (0.02 M iodine in pyridine/tetrahydrofuran/water) were purchased from Glen Research (Sterling, VA). Phosphoramidites and 5-ethylthio-1H-tetrazole were dissolved at 0.25 M in a mixture of 50% 3-methoxypropionitrile and 50% glutaronitrile (Sigma, St. Louis, MO). The solvents were dried for two days on molecular sieves before use. Synthesis-grade anhydrous acetonitrile and high-purity dichloromethane and dichloroacetic acid were purchased from Sigma. The detritylation solution used was 2.5% dichloroacetic acid in dichloromethane. Only 6 pl of phosphoramidite monomers and tetrazole were spotted on the slide using the inkjet print head; all other reagents were added 1 ml at a time using PTFE solenoid valves.
The internal atmosphere of the platform was flushed with dry nitrogen for 30 min at 60 l/min before synthesis is initiated. Humidity was monitored using a capacitance hygrometer (Model 4187, Control Company, Friendswood, TX) and oxygen was measured using an Oxy-Plus electrochemical device (Brandt Instruments, Slidell, LA). The nitrogen stream continued at 35 l/min until synthesis was complete. Slides were washed three times with acetonitrile. The reagents were blown off the edge of the slide with a reciprocating motion from a manifold of nitrogen jets.
Each cycle of synthesis includes (1) printing, (2) washing, (3) oxidation, (4) washing, (5) detritylation, and (6) washing. The first step of the reaction consists of phosphoramidite monomer printing followed by tetrazole printing, followed by a 2 min incubation. This first 3' base was reprinted to ensure a high density of oligonucleotide synthesis. Oxidization and detritylation steps were also carried out for 2 min each.
After reporter synthesis was completed, the microarray slides were removed from their slide holder and rinsed with acetonitrile and then 95% ethanol. The base-protecting groups were removed by a 105 min incubation in an aqueous solution of 13% ammonium hydroxide and 20% methylamine in a sealed container at room temperature. The deprotected slides were rinsed five times with de-ionized water, dried, and stored in a desiccator.
Images of droplets
Images were acquired using a Zeiss Axiophot stereomicroscope equipped with a 5 × Plan-Neofluor objective and standard 10 × planar eyepieces, connected to a Roper Scientific cooled-CCD digital camera (1,392 × 1,040 resolution, pixel size 6.45 × 6.45 μm, firmware revision 8.69).
RS Image Acquisition settings were as follows: exposure type, normal; exposure mode, timed; automatic exposure with intensity target of 2,000 and upper limit exposure time of 1 sec; full-CCD mode with speed 20,000 kHz; gain equal to 1; no shutter delay; neutral brightness, contrast, and gamma, with white auto balance and 5%-95% scaling.
Hybridizations were carried out using labeled targets listed in Table 1, purchased from IDT (Coralville, IA), and diluted to a 100 nM final concentration with DIG Easy Hyb buffer (Roche Molecular Biochemicals, Indianapolis, IN). Forty microliters of hybridization solution was placed on the microarray and a coverslip carefully applied. The slide was placed in a hybridization chamber and incubated for 1 h incubation at temperatures indicated in the figure legends. The coverslips were removed and the microarrays were washed for 15 min in 1 × SSC + 0.5 % SDS buffer followed by a 5-sec wash in 0.1 × SSC + 0.1% SDS at room temperature. The microarrays were dried under a nitrogen stream. Microarray images were collected using 10 μm resolution, 60-90% laser power, and 80-90% PMT gain on a ScanArray 5000 laser scanner (Packard BioScience, Meriden, CT) and saved as 16-bit TIFF images. The excitation/emission filter settings used were 532 nm/546 nm for measuring Cy3 and 633 nm/670 nm for Cy5 and Bodipy. The fluorescence intensities were determined using Dapple image-analysis software [32]. Dapple output files, annotated with feature names and sequences, can be found in Additional data file 3. Microarrays were stripped by washing in 20 mM sodium hydroxide for 2 min, followed by five washes in de-ionized water.
Labeling and quantification of targets
Nucleic acid and and fluorophore dye concentrations were determined by spectrophotometric measurement of absorbance at λ = 260 nm for nucleic acid and at the excitation peak λ = 550 nm for Cy3, λ = 650 nm for Cy5) for the dye reporter. Concentrations were calculated using appropriate extinction coefficients (A260 = 50 μg/ml dsDNA at 1 OD, A260 = 33 μg/ml ssDNA at 1 OD, A550 = 150,000 M Cy3, A550 = 250,000 M Cy5), and samples were normalized for each hybridization to provide equimolar amounts of each fluorophore.
Direct chemical labeling
Oligoarrays of 20 mers were printed as described in the synthesis section of the Materials and methods, except that reporters contained from 0 to 10 dGMP moieties distributed along the length of the molecule. The position of reporters containing different numbers of dGMPs were randomized before printing. ULYSIS nucleic acid labeling kit (Molecular Probes) was used to label the N-7 position of the dGMPs by a modification of the instructions recommended by the manufacturer: 70 μl of degassed Universal Labeling System (ULS) reagent containing AlexaFluor-647 with DMSO was diluted 1:100 in Component C buffer. The reaction mixture was placed on top of the reporters on the oligoarray slide and incubated under a coverslip at 80°C for 15 min. After incubation, the coverslip was removed and the slide was incubated in 2 × SSC, 0.2% SDS for 10 min. Slides were thoroughly washed with de-ionized water, blown dry with compressed nitrogen, and scanned on a ScanArray 5000 microarray scanner using laser power and PMT gain settings of between 65% and 85%.
Preparation of genomic DNA
Ninety-four different barcoded yeast deletion strains were grown in rich medium containing 100 mg/L G418 (geneticin) at 30°C. Three additional cultures containing only strain YPL110C, YPL111W or YPL112C were grown using identical conditions. After 4 h, the cell density was determined by the OD600. Two milliliters (approximately 5 × 107 cells) of each population were used to extract genomic DNA.
Each set of cells was pelleted, washed with diH2O, pelleted again and resuspended in 200 μl breaking buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris pH 8, 1 mM EDTA pH 8). Two hundred microliters of 24:1 phenol:chloroform and 200 μl siliconized glass beads were added, and the cells were lysed by agitation. Two hundred microliters of TE buffer (10 mM Tris-Cl, 1 mM EDTA, pH 7.5) was added and cell debris removed by centrifugation at 13,000 g for 5 min. The aqueous phase was collected and nucleic acids were precipitated by the addition of 1 ml 100% ethanol at room temperature followed by three minutes of centrifugation at 13,000 g. The pellet was resuspended in 400 μl TE. Contaminating RNA was hydrolyzed in the presence of 30 μg RNase A for 5 min at 37°C. Genomic DNAs were precipitated by the addition of 10 μl 4 M ammonium acetate and 1 ml 100% ethanol at room temperature. After centrifugation, the DNA pellets were resuspended in 100 μl of TE and the concentration determined by absorbance at 260 nm/280 nm.
PCR amplification
Barcode tags from the genomic DNA were amplified and labeled by PCR. Reactions were 100 μl in scale and contained 20 mM dNTPs, 150 mM MgCl2, 1 × Fermentas Taq DNA polymerase buffer, 100 μM each of Cy3 or Cy5 conjugated forward and reverse primers, 1 μg of the genomic DNA and 3.75 units of Taq DNA polymerase. The enrichment reactions were spiked with 100 ng each of DNA from barcode strains YPL110C, YPL111W and YPL112C. Mixes were heated to 94°C for 3 min, followed by 30 cycles of 94°C for 15 sec, 50°C for 15 sec, and 72°C for 30 sec. Reactions were finished by a final incubation at 72°C for 3 min. PCR products were purified using the Qiagen nucleotide removal kit following the manufacturers' recommended protocol.
POSaM barcode oligoarrays
Each barcode array slide contained a grid of registration features containing sequences complementary to a labeled 20-base control oligonucleotide. Their hybridization is used to verify feature size, shape and uniformity, as well as to aid in the registration of grids used by the spot-finding program, Dapple. No cross-hybridization was observed between the barcode reporter sequences and the control oligonucleotide. The barcode reporters consisted of 38-base inkjet synthesized oligonucleotides. The 'perfect match' reporters contain 20 bases complementary to either the Watson or Crick strands of the identifying barcode plus 18 bases of primer sequence. 'Mismatch' reporters were also synthesized, containing four evenly spaced mutations. Each sequence was represented in quadruplicate in the array; thus each barcode PCR product could hybridize to eight perfect-match reporters.
Probing of POSaM barcode oligoarrays
The purified Cy3/Cy5-labeled PCR products were quantified at OD550 (Cy3) or OD650 (Cy5) and 7 pmol of each labeled PCR product was used to probe the barcode oligoarrays. The PCR products were combined as in a 'dye-flip' experiment: one solution combining Cy3 reference reaction with Cy5 enrichment reaction and the other solution combining Cy5 reference reaction with Cy3 enrichment reaction. The solutions were concentrated by precipitation and resuspended in 47 μl 1 × DIG Easy Hyb buffer (Roche), 3 μl yeast tRNA (10 mg/ml), and 10 μl sheared salmon sperm DNA (10 mg/ml). The resulting mix was heated to 90°C for 1 min, placed on ice for 1 min, and kept at 42°C until use.
POSaM oligoarray slides were prehybridized in a 3 × SSC (450 mM sodium chloride, 45 mM sodium citrate), 0.1% SDS, 0.1 mg/ml sheared salmon sperm DNA for 30 min at room temperature. The oligoarrays were washed in diH2O, dried, and hybridized with the labeled probe mixes for 1 h at room temperature. Hybridized slides were then washed with increasing stringency: first with 1 × SSC (150 mM sodium chloride, 15 mM sodium citrate), 0.2% SDS at 55°C for 5 min, then with 0.1 × SSC, 0.2% SDS at room temperature for 5 min, then with 0.5 × SSC at 45°C for 5 min, and finally with two washes of 0.1 × SSC at room temperature. Slides were dried with compressed air and scanned on a ScanArray 5000 with laser power and photomultiplier gain settings at 85%.
Additional data files
Additional data available with the online version of this article consists of PDF files containing instructions for assembling (Additional data file 1) and using (Additional data file 2) the POSaM and a zip file (Additional data file 3) that contains the raw microarray fluorescence data used to generate the results shown in Figures 6, 7, 8, 9, 10, 11.
The authors thank David Shteynberg, Brett Kotschwar and Matthew von Bargen for software development help, Stephen Mordue and Avid Shiau for testing, and Eva Aw for technical assistance. This work was funded by NIH grants HG01713 and HG02931.
Authors' Affiliations
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Child's Play
Men in Black: International
The Men in Black have always protected the Earth from the scum of the universe. In this new adventure, they tackle their biggest, most global threat to date: a mole…
Toy Story 4
What if a child from another world crash-landed on Earth, but instead of becoming a hero to mankind, he proved to be something far more sinister?
The Intruder
Dark Phoenix
The X-Men face their most formidable and powerful foe when one of their own, Jean Grey, starts to spiral out of control. During a rescue mission in outer space, Jean…
The Aftermath
The Hummingbird Project
Godzilla: King of the Monsters
The new story follows the heroic efforts of the crypto-zoological agency Monarch as its members face off against a battery of god-sized monsters, including the mighty Godzilla, who collides with…
The Secret Life of Pets 2
The Poison Rose
Avengers: Endgame
After the devastating events of Avengers: Infinity War, the universe is in ruins due to the efforts of the Mad Titan, Thanos. With the help of remaining allies, the Avengers…
Godzilla: King of the Monsters
The new story follows the heroic efforts of the crypto-zoological agency Monarch as its members face off against a battery of god-sized monsters, including the mighty Godzilla, who collides with…
Captain Marvel
The story follows Carol Danvers as she becomes one of the universe's most powerful heroes when Earth is caught in the middle of a galactic war between two alien races….
Dark Phoenix
The X-Men face their most formidable and powerful foe when one of their own, Jean Grey, starts to spiral out of control. During a rescue mission in outer space, Jean…
Men in Black: International
The Men in Black have always protected the Earth from the scum of the universe. In this new adventure, they tackle their biggest, most global threat to date: a mole…
Pokémon: Detective Pikachu
Game of Thrones
Seven noble families fight for control of the mythical land of Westeros. Friction between the houses leads to full-scale war. All while a very ancient evil awakens in the farthest…
John Wick: Chapter 3 – Parabellum
Toy Story 4
The Secret Life of Pets 2
Fast Color
A woman is forced to go on the run when her superhuman abilities are discovered. Years after having abandoned her family, the only place she has left to hide is…
Jurassic World: Fallen Kingdom
Three years after the demise of Jurassic World, a volcanic eruption threatens the remaining dinosaurs on the isla Nublar, so Claire Dearing, the former park manager, recruits Owen Grady to…
Black Mirror
A contemporary British re-working of The Twilight Zone with stories that tap into the collective unease about our modern world. Over the last ten years, technology has transformed almost every…
The Sun Is Also a Star
The Flash Season 2 Episode 11
When Cisco gets a vibe of Eobard Thawne, Barry and the team don't believe it. But, after an attack at Mercury Labs, Christina McGee confirms that the Reverse Flash is…
The Upside
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The Guardians must fight to keep their newfound family together as they unravel the mysteries of Peter Quill's true parentage.
Zindagi Na Milegi Dobara
Three friends who were inseparable in childhood decide to go on a three-week-long bachelor road trip to Spain, in order to re-establish their bond and explore thrilling adventures, before one…
Let It Shine
A dance musical that explores the juxtaposition of several segments of 1980s life and society in New York: the ball culture world, the rise of the luxury Trump-era universe and…
Dear John
The Conjuring
Pirates of the Caribbean: On Stranger Tides
Wu-Tang Clan: Of Mics and Men Season 1 Episode 2
Wu-Tang Clan's underground rap single blows up and business moved fast. Despite their success, things were going on back home in the projects that affected their friends and family –…
Game of Thrones Season 8 Episode 2
Wally Got Wasted
Three friends accidentally kill a scum bag, then have to pretend he is still alive to cover up their crime.
In the summer of 2015, former US Marine and world record weightlifter Matt "Kroc" Kroczaleski was publicly outed as being transgender. The reaction was universal: her sponsors abandoned her, she…
Jamario, Jaquan, Jailen, and Teague are teammates on the J.O. Johnson High School wrestling team in Huntsville, Alabama. Led by their passionate coach, they are trying to qualify for the…
Ghost Squad
It's 1996 and 4th grader, Cecil Stevens, is reminded of his terrible lisp every time he says his name. To avoid social travesty, Cecil decides to change his hard to…
The documentary follows one woman's quest to overcome anxiety, depression, and opioid addiction through the use of psychedelic medicines.
Doctor Who: The Day of the Doctor
In 2013, something terrible is awakening in London's National Gallery; in 1562, a murderous plot is afoot in Elizabethan England; and somewhere in space an ancient battle reaches its devastating…
Little Mix: Glory Days – The Documentary
A documentary following a year in the life of Little Mix. Shows the positives and negatives of being the worlds biggest Girlband!
Sleeping Beauty: A Gothic Romance
Matthew Bourne choreographs this version of Tchaikovsky's ballet performed at Sadler's Wells Theatre. Bourne sets the first part of the story in 1890, the year in which Tchaikovsky completed his…
The Shawshank Redemption
Pink Floyd: Pulse
Pulse (stylized as P•U•L•S•E) is a Pink Floyd concert video taken from the October 20, 1994 concert at Earls Court Exhibition Centre, London, in The Division Bell tour, originally released…
The Mountain II
Crossing the Bridge
An unusual love story about two young people which is set in the divided city of Mostar in Bosnia and Herzegovina.
The Godfather
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In a mysterious and remote snowy outpost, Matt and Potter share a Christmas meal, swapping creepy tales of their earlier lives in the outside world, but are both men who…
A-Bombs Over Nevada
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Determined to keep Annabelle from wreaking more havoc, demonologists Ed and Lorraine Warren bring the possessed doll to the locked artifacts room in their home, placing her "safely" behind sacred…
The Prince's Trust Rock Gala 1990
Filmed at the 1990 performance of Prince Charles's annual charity gala, this home video captures the regal rock 'n roll event in all it's glory. The concert features performances by…
Saz – The Key Of Trust
A documentary by Stephan Talneau
The Bad, the Black and the Beautiful
Cmd + Ctrl
During a quiet evening at home a couple test a prototype biohacking technology, but what begins as playful experimentation turns sinister as neither are aware of the dangers it poses.
Rolling Stones – 50 Years On Video – Red Edition
The Rolling Stones – 50 Years on Video [FROM THE VAULTS FTV-002DVD1/2] Video: MPEG-2 NTSC 720×480 16:9 Audio: Dolby digital stereo Duration: Approx.151 min.
A woman receives the chance to relive the life of her younger self, at a point in her life when the pressures of adulthood become too much for her to…
Pet Sematary
Child's Play
When he was 14, Smith drowned in Lake St. Louis and was dead for nearly an hour. According to reports at the time, CPR was performed 27 minutes to no…
How to Answer the Fool
Produced as a cinematic film, How to Answer the Fool was adapted for use in small group and Sunday School classes, making it a useful tool for individual or group…
WWE Stomping Grounds
A multigenerational family saga centered around one woman who, having recently relocated and intent on revitalizing her marriage, finds herself going head to head with some of the most powerful…
A group of high school students navigate love and friendships in a world of drugs, sex, trauma, and social media.
A grieving teen finds an unexpected connection with two classmates at her new high school after they all land in the same Shoplifters Anonymous group.
Fresh out of prison, world-class thief Daisy "Jett" Kowalski is forced back into doing what she does best by dangerous and eccentric criminals determined to exploit her skills for their…
Legends of the Deep
Celine and Fabien Cousteau use cutting-edge tech to hunt for a long-lost shipwreck in the Bermuda Triangle, and if their top-secret clues are right, they might uncover new evidence that…
The Last 24
Follow determined investigators working the most shocking murder cases as they reconstruct the tragic final hours in the victim's life, in order to find their killer and crack the case.
Malibu Rescue: The Series
On the heels of Junior Rescue training, Team Flounder returns to brave the beach in a series of thrilling saves and lighthearted laughs.
Arthur and the Children of the Round Table
American Princess
When she discovers her fiance is cheating on her hours before their wedding, Manhattan socialite Amanda runs off to join a Renaissance fair as the latest wench-in-training.
Señoras del HAMPA
Perpetual Grace LTD
James, a disgraced firefighter turned grifter, seizes a chance, illicit opportunity to reverse his life's worsening course as he attempts to prey upon Pastor Byron Brown, who turns out to…
A young Victoria "Vic" McQueen discovers she has the power to unlock portals that help her find lost objects. As she delves further into this hidden universe, she encounters many…
Arthdal Chronicles
In a mythical land called Arth, the inhabitants of the ancient city of Arthdal and its surrounding regions vie for power as they build a new society.
How to Sell Drugs Online (Fast)
Swamp Thing
CDC researcher Abby Arcane investigates what seems to be a deadly swamp-born virus in a small town in Louisiana but she soon discovers that the swamp holds mystical and terrifying…
When They See Us
This docuseries exposes the breakdown of the U.S. criminal justice system during the Central Park Five case where five teenagers of color were convicted of a rape they did not…
Wentworth Season 7 Episode 5
The Handmaid's Tale Season 3 Episode 6
Legion Season 3 Episode 1
David gains a following.
Big Little Lies Season 2 Episode 3
Good Witch Season 5 Episode 4
Fear the Walking Dead Season 5 Episode 4
Fear the Walking Dead Season 5 Episode 3
Harrow Season 2 Episode 7
Baskets Season 4 Episode 1
Susan nearly kills Ronald Reagan.
Baskets Season 4 Episode 2
Chip picks up Pedialyte.


text:
Let $A$ be a finite abelian group, and let
$ \psi : A \times A \to \mathbb{Q}/\mathbb{Z} $
be an alternating, non-degenerate bilinear form on $A$. Maybe I should say what I mean by these words; bilinear means it is linear in each argument separately; alternating means that $\psi(a,a) = 0$ for all $a$; non-degenerate means that, if $\psi(a,b) = 0$ for all $b$, then $a$ must be $0$.
Why must $A$ have square cardinality?
I believe it will follow from the following theorem in Linear algebra:
Theorem. Let $V$ be a finite dimensional vector space over a field $K$ that has an alternating, non-degenerate bilinear form on it (from $V \times V \to K$). Then dim $V$ is even.
My idea was to proceed as follows: If the size of $A$ is not square, then for some prime $p$, $A(p)$ is not square, where $A(p)$ means the $p$-primary part of $A$. The original $\psi$ induces a map on $A(p)$ that is non-degenerate, alternating and bilinear. I then wanted to say that $A(p)$ is an $\mathbb{F}_p$-vector space, and then applying the theorem I am done, but this is not true, e.g, $\mathbb{Z}/25\mathbb{Z}$ is not an $\mathbb{F}_5$-vector space.
Any pointers anyone?


text:
Chai Concentrate
From: Munchin with Munchkin
Quick Facts
Servings: 5
Prep Time: 10 min.
Cook Time: 25 min.
Total: 35 min.
Kris Mullen
Nothing warms you up like a cup of this fragrant Indian beverage. You can also add it to baked goods to make chai spiced cookies, cakes, or even pie.
- 5 cups water
- 10 bags of black tea (try a mixture of orange pekoe and English breakfast)
- 2 cinnamon sticks
- 1 vanilla bean, sliced in half
- 1/2 cup brown sugar
- 12 whole cloves
- 9 cardamon pods
- 1/2 tsp freshly ground nutmeg
- 2 star anise pods
- 1/2 tsp black peppercorns
- 8 corriander seeds
- 1/2 tsp ground allspice
- 1 Tbs orange zest
- 1 inch piece of ginger, sliced into discs
- 1 Tbs honey (optional)
- In a large pot, bring water to a rapid boil. Add tea bags, spices, orange zest, and ginger. Reduce heat, cover, and simmer for 20 to 25 minutes.
- Remove from heat, stir in honey and let stand 10 minutes.
- Line a sieve with cheesecloth and strain concentrate. Discard spices.
- Store concentrate in a sealable container in the fridge for up to two weeks.
- To make chai tea, combine 1/3 cup of concentrate with warm milk (or soy/nut milk).
- Concentrate can also be mixed in with a number of recipes. Try mixing it with cookie batter for chai spiced cookies or mix with seltzer water for a chai soda!
Nutrition Facts
Calories 144
Calories from Fat 7 (5%)
(2%)Total Fat 1g
(1%)Saturated Fat 0g
Polyunsaturated Fat 0g
Monounsaturated Fat 0g
(0%)Cholesterol 0mg
(1%)Sodium 35mg
(10%)Potassium 367mg
Total Carbohydrate 37g
(21%)Dietary Fiber 5g
Sugars 25g
Sugar Alcohols 0g
(2%)Protein 1g
* Percent Daily Values are based on a 2,000 calorie diet.
Nutrition facts are calculated by a food expert using nutritional values provided by the USDA for common products used as recipe ingredients. Actual nutritional values may differ depending on the amounts or products used and can be affected by cooking methods.
Copyright © 2019 Healthnotes, Inc. All rights reserved. www.healthnotes.com
Read our healthy recipe definitions.
Learn more about Healthnotes, the company.
The information presented here is for informational purposes only and was created by a team of US–registered dietitians and food experts. Consult your doctor, practitioner, and/or pharmacist for any health problem and before using any supplements, making dietary changes, or before making any changes in prescribed medications. Information expires December 2019.


text:
Westboro Baptist Church to Protest Okla. Liquor Store
Days after the death of their excommunicated founder, the Kansas-based Westboro Baptist Church has announced plans to picket an Oklahoma liquor store over a sign they put up recently.
On Twitter, Westboro Baptist commented that they intended to picket the Moore Liquor Marquee on Saturday, April 5.
The sign, posted Tuesday, read "Fred Phelps 1929-2014" and added at the bottom "Champagne 10% off! Not a coincidence."
Bryan Kerr, owner of Moore Liquor, told The Christian Post that Phelps was "a man whose ideas and actions spread more hate and vitriol than nearly anyone else I can think of in modern-day America."
"Although I am not normally one to speak ill of the recently departed, I felt this particular event deserved some celebration since it brings us one step closer to the Westboro Baptist Church becoming a footnote in our religious history books," said Kerr.
Kerr also told CP that the overall response to his sign was largely positive, adding that he did not expect to garner such immense compliments from across the country.
"I was unaware that the sign would move so many people to action. We've had calls, posts and emails from all over the country," said Kerr.
"Most people just wanted to say they appreciated the sentiment or that they were 'with us.' There have been a few negative comments, mostly regarding my lack of respect for a deceased person and his grieving family. But those have been few and far between," he added.
Last week, the 84-year-old Fred Phelps died in hospice care in Kansas. Unaffiliated with any Baptist denomination, Phelps' Westboro Church continues to organize protests and pickets.
Westboro has garnered a reputation for its hateful protests at funerals, high schools and major events like the Super Bowl.
Members of the church, which is mostly made up of Phelps' family, are known for showing up with signs with messages like "Thank God for dead soldiers" and "God hates fags."
The church has also claimed that the 9-11 terrorist attacks were proof of God's judgment on the United States for allowing things like homosexuality and abortion.
Regarding the prospect of a Westboro protest outside Moore Liquor, Kerr told CP that he was "very pleased" with the their intention of paying him a visit.
"If it keeps them from spending one extra minute at a soldier's funeral, it's worth any headache they may cause at Moore Liquor," said Kerr.
"I think this particular protest will help shuffle them a bit closer to being the cartoon characters that they've been slowly becoming for years."


text:
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Settle Your Claim
If you're 50-plus with a claim at least 180 days old, an alternative to monthly time-loss benefits is a structured settlement agreement.
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Find L&I approved training programs and information about Option 2: Developing your own training plan.


text:
Minimum wage laws are regarded by economists of all stripes as among the least efficient tools for boosting the welfare of those at the lower end of the earnings spectrum, inevitably harming many of those they are intended to help.
This leads some to conclude that politicians who champion increasing the minimum wage must be demagogues working for partisan advantage. In reality, most politicians simply don't understand economics. In fact, there is research to support this more generous view.
In 2011, scholars J. Brian O'Roark and William C. Wood published a study on the link between minimum wage votes by members of Congress and their formal educations. The study, "Determinants of Congressional Minimum Wage Support: The Role of Economic Education," concluded that "members who majored in economics as undergraduates were less likely to vote for the minimum wage increase than their colleagues. No other major had a consistent influence."
O'Roark and Wood call for "deference" to economists on this topic, writing that "the minimum wage debate is a particularly political one, but just as the opinions of doctors on medical issues are deserving of special consideration, we believe that the opinions of the most economically literate on the minimum wage should be carefully considered by policymakers and the public."
Another lens through which to examine this problem looks at what politicians do rather than what they say. Mackinac Center for Public Policy Research Associate Jarrett Skorup recently documented the apparent hypocrisy of some politicians who publicly call for increased minimum wages while also employing office interns at a wage of $0.
Politicians are not the only hypocritical actors in this play. It is reminiscent of a California lawsuit filed several years ago by ACORN, a political agitprop group with strong ties to the Service Employees International Union. ACORN promoted not just minimum wage hikes, but even more burdensome "living wage" mandates.
The lawsuit sought to exempt ACORN, however, from paying its own members the wages it wanted mandated on everyone else. The group defended itself by stating the obvious case that other businesses could make as well. As ACORN lawyers wrote in their brief, "[T]he more that ACORN must pay each individual outreach worker … the fewer outreach workers it will be able to hire."
That is exactly the type of insight we would expect from someone with knowledge of the economic science. Like the congressmen studied by O'Roark and Wood, perhaps these champions of higher wage mandates for "thee but not me" had also taken college economics courses.
Minimum wage laws have been shown to eliminate job opportunities and cost many of the least skilled workers their jobs. Politicians who support increasing wage mandates may be ignorant of the economics at work here, or may simply be willing to sacrifice low-wage workers to gain political advantage. The public should correct them by rejecting these policies outright.
Permission to reprint this blog post in whole or in part is hereby granted, provided that the author (or authors) and the Mackinac Center for Public Policy are properly cited. Permission to reprint any comments below is granted only for those comments written by Mackinac Center policy staff.
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text:
Cowboys quarterback Tony Romo will miss Organized Team Activities after having surgery to remove a cyst from his back.
Romo, however, says there's nothing to worry about, and that the procedure was fairly minor.
"If this was the regular season and I had to play next week, I could," Romo told the Dallas Morning News. "This is just about being smart. That's why I did it now. This will have no effect on training camp. No way will it have an impact. And I still think there's a good chance I'm on the field for minicamp."
Although it hadn't been reported previously, Romo had the cyst removed a month ago. He said the surgery was simple enough that his wife could drive him home that day, and he was playing with their son that evening.
"It was a pretty routine afternoon,'' Romo said. "I was back at the [Cowboys] complex the next day to go through what we would do in the teaching sessions and the throwing sessions this off-season. . . . It was not much of anything, I just went in and had them look at it. It was more of an ache than anything. I just thought, 'well, it's something I'd rather not feel.' I'd rather be 100 percent, not 96 percent. It's done and it's made me feel better."
And while Romo isn't feeling enough to get to work now, he's expecting to be as good as ever before the season starts.


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Simple and delicious, this sensational recipe for Asian Sesame Ginger Salmon is sweet, fragrant, and full of fabulous flavour. Low-cal and carb-free, the salmon is marinated in an aromatic mixture of sesame oil, rice vinegar, brown sugar, soy sauce, ginger and garlic, then chilled for at least 30 minutes to allow the flavours to work their magic. While the salmon is marinating, a simple sauce of honey, sesame oil, soy sauce, minced ginger and sesame seeds can be whisked together and gently warmed on low heat, ready to be served over the cooked salmon with a final garnish of sesame seeds and chopped green onions.
Asian Sesame Ginger Salmon
Serves 4
4 organic salmon filets, about 2-pounds
1/4 cup olive oil
2 tbsp toasted sesame oil
2 tbsp rice vinegar
2 tbsp brown sugar
2 tbsp soy sauce
2 garlic cloves, minced
1 tbsp freshly grated ginger
2 tbsp sesame seeds
4 green onions, finely sliced for garnish
Honey, Ginger & Sesame Sauce:
1/4 cup honey
1 tsp toasted sesame oil
1 tsp soy sauce
1/2 tsp freshly grated ginger
1/2 tsp sesame seeds
In a medium bowl, combine the olive oil, sesame oil, soy sauce, garlic, ginger, vinegar, brown sugar and whisk well until combined. Add the salmon, cover and refrigerate to marinate, for 30 minutes. Meanwhile, prepare the sauce by combining each of the ingredients in a small bowl and whisk until smooth.
Preheat oven to 425°F and line a baking sheet with aluminum foil. Place the salmon on the baking sheet and bake for 10-12 minutes, until the centre is just done. If you like your salmon more well done, simply bake as long as you wish. To serve, spoon some of the sauce over top of the salmon and garnish with a sprinkle of sesame seeds and flurry of sliced green onions.


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The Jetstream is used to train the all important Observers of the Fleet Air Arm who eventually become the tacticians of our Sea King Mk7 ASaC (Airborne Surveillance and Control), Merlin (anti-submarine) and Lynx (anti-surface) helicopters. To facilitate this training, the Jetstream is fitted with a nose mounted radar and two navigation consoles for the student and instructor. The jetstream is a fixed wing aircraft powered by twin gas turbine engines and with spare passenger capacity. There are two variants in service, T2s, which are used in the observer training role and T3s, which are operated by Heron flight at Yeovilton for operational support and communications.
|2 x 940hp Turbomeca 16D turboprops
|1 or 2 pilots
More Detail: 750 Squadron


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Each participating company has generously donated towards the prize package listed on the Spellbinders Blog. A total of two identical prize packages, one for each day, November 18 and 19 will be given away! To enter, just leave a comment on a participating blog! Comments in the first 48 hours of the post are eligible. One commenter each day will be randomly selected as the winner. Winners will be announced on November 22, on the Spellbinders Blog!
For this project I used my Spellbinders Grand Labels One to frame the image of my stamped Santa. I had seen a paper poinsettia wreath on the Martha Stewart website and was inspired to make my own interpretation thereof using Spellbinders die templates, cardstock, patterned paper, glitter and stamps. Here is a list of supplies you will need:
One winner will be chosen from all the comments on all the blogs participating today. Comments made in the first 48 hours of this post will be eligible to win. Winner's names will be published on the Spellbinders Blog on November 22, and they will have 48 hours to reply to Spellbinders to claim the prizes.


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Are you one of those people who has been inspired to learn more about your own family history after watching television documentaries about the family backgrounds of well known people?
Are you an avid reader of biographies and autobiographies?
Have you been told that you are related to a famous or otherwise important person, or even a notorious one?
I am one of those people who has no famous or illustrious ancestors, as far as I am aware. No-one in my family is a "celebrity". Most of the people you may think of as celebrities are probably unfamiliar names to me, especially as I rarely watch television. I do like watching documentaries about historical events and the unfolding of interesting lives. Some of these are now available online or on DVD.
Ongoing series about celebrity genealogy include these:
The BBC also has a useful family history website
For those of you in the United States, keep your Wednesdays free between February 10 and March 3, from 8-9pm ET for a PBS series called Faces of America. It is presented by Harvard scholar Henry Louis Gates, Jnr.
There are also many magazines and books on genealogical topics, however I have been able to do most of my research without using any of these. Most of the information I have found has been freely available, both online and by directly contacting people who might know more than me.
I have recently purchased a copy of the November 2009 edition of a UK magazine called Your Family History. It was rather expensive to buy on a one-off basis here in Australia but I wanted to have a copy at home for reference purposes.
Well, that's all for now. Good viewing, good reading, good research, and good luck!


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Jackson County could get as much as $3.5 million in a one-year extension of federal timber payments passed by both houses of Congress this week.
The extension was included in a bill that averts a shutdown of the federal helium reserve. If signed by President Obama as expected, it will mean about $100 million for 33 Oregon counties.
Rocky McVay, of the Association of O&C Counties, said the exact amount Jackson County would receive is not known, but he expects it to be close to the $3.5 million the county received in 2012-13. Those funds likely would be paid in January or February 2014.
"The caveat to that is we don't know what they're going to do with the budget," McVay said.
Jackson County Board of Commissioners Chairman Don Skundrick said most of the money would go into the county's rainy-day fund since it's a one-time infusion.
Josephine County would see about $4.4 million to be used for law enforcement in the next fiscal year beginning July 1, 2014, Rosemary Padgett, chief financial officer for the county, told the Grants Pass Daily Courier.
"The shoe has yet to drop. We keep limping along here," said Josephine County Commissioner Simon Hare after getting the news. "We're still going to have more significant reductions. We're almost at a breaking point at the jail."
U.S. Rep. Greg Walden, R-Ore., said the measure is a short-term solution, but that broader, long-term solutions are needed to provide relief in rural communities.
"That help is a lifeline, not a lifeboat," Walden said of the extension from the House floor. "The status quo of asking Uncle Sam for a check year after year is simply not sustainable. However, managing our federal forests, generating jobs and revenue; that's sustainable."
Walden added he hopes the Healthy Forests for Healthy Communities Act, passed last week by the House, would provide that long-term solution, utilizing forest management that would provide tax revenue for local schools and law enforcement.
"We need to help our forested communities get back to self-sufficiency and self-reliance, get people back to work in the woods," Walden said. "The clock's ticking. It's time to get this done."
The Health Forests act would place the roughly 2.7 million acres of so-called O&C land into two trusts, with roughly half of it managed for conservation while the remainder would focus on sustainable timber production to help fund county coffers.
Conservation groups have decried the proposal as an attempt to privatize federal forestland and warn of a return to clear-cuts.
The extension of timber payments to cash-strapped counties was included in the Responsible Helium Administration and Stewardship Act, or HR 527.
The bill was passed by the House Wednesday and the Senate on Thursday.
The House's Committee on Natural Resources said in a news release that HR 527 is intended to preserve the U.S. helium supply by preventing the closure of the Federal Helium Reserve and reducing the federal deficit by $90 million over 10 years.
The Federal Helium Program, which provides about 42 percent of the nation's helium from a storage site in Texas, was set to shut down Oct. 7 unless lawmakers intervened. Closing the reserve could cause a worldwide helium shortage.
The timber payments amendment was included by U.S. Sen. Ron Wyden, D-Ore., who is chairman of the Senate's Energy and Natural Resources committee, which has jurisdiction over the National Helium Reserve.
Reach Mail Tribune reporter Ryan Pfeil at 541-776-4468 or by email at firstname.lastname@example.com. The Associated Press and Grants Pass Daily Courier contributed to this report.


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PHOENIX – During his 2,097-yard MVP blitzkrieg last season, Vikings star Adrian Peterson ran with the unbridled tenacity for which he's always been known, dishing out vicious shots as often as he absorbed them.
Peterson has excelled with that Pamplona bull style. But now, with the NFL in heated debate over a rule proposal that would penalize ball carriers for delivering contact with their heads, it's worth wondering whether some of Peterson's most impressive and physical runs could now occasionally result in penalties against the Vikings.
On Tuesday at the NFL's annual meetings at the Arizona Biltmore, debate continued over a proposed rule that would require a 15-yard penalty to be assessed if any ball carrier, while outside the tackle box and more than 3 yards down field, "initiates forcible contact by delivering a blow with the top/crown of his helmet against an opponent."
It's a pitch that has been greeted by many coaches as well-intentioned from a safety standpoint but perhaps misguided if enforced too aggressively.
The Vikings have made it clear they are wary of the rule. Owner Zygi Wilf spoke up twice during debate Tuesday morning. Coach Leslie Frazier, while noting the admirable safety objective, also raised an eyebrow at how to reasonably make such a rule work.
"I think it makes it tough for the running backs," Frazier said. "It's such an instinctive motion to duck that head to try to protect yourself.
"So I don't know what's going to happen, whether this will pass or not. But I think it puts those backs in a vulnerable state if they can't lower their pads. And the only way to lower your pads is to get your head down."
For the rule change to be adopted, it will require three-quarter approval in a final vote that has been tabled until Wednesday. At present, however, vocal opposition has been evident.
Frazier said he thinks the rule's intent to reduce head injuries could wind up having an adverse effect on player safety if ball carriers begin to run more upright without ability to protect themselves legally by ducking into collisions.
"When those defenders are coming at your legs, if you don't protect yourself and you don't get your pads down, now you run the risk of lower body injuries," Frazier said.
Yet Rams head coach Jeff Fisher, who doubles as an active member of the competition committee, has made it clear that if the rule were adopted, officials would be instructed to look for a very specific infraction.
"We want to bring the shoulder back to the game," Fisher said.
"We all know the helmet is a protective device. And it's not designed to be used like it's been used as of late."
Dean Blandino, the NFL's vice president of officiating, said part of the research that went into the rule proposal involved reviewing every play from Week 16 last season. In those 16 contests, Blandino said there were 34 instances of helmet-to-helmet contact between ball carriers and tacklers but only five that would have drawn penalties under the new rule.
"There is going to be helmet-to-helmet contact between a runner and a tackler," Blandino said. "That is not what this rule is designed to take out.
"It is where one player lowers the head and delivers a blow with the crown, the top of the helmet, lines up a player and hits him when we feel he has options to do something else."
Still, even with the competition committee and officials trying to zero in on "the obvious foul," coaches worry the speed of the game and the fluctuating discretion of each officiating crew could create too much confusion from week to week, series to series.
As of Tuesday morning, Pittsburgh coach Mike Tomlin didn't know whether he'd advise the Steelers to check the "For" box on their ballot or the one marked "Against."
"I'm interested in more discussion to be honest with you," Tomlin said. "I think that's the charge.
"I think it's easy to say we want to eliminate those type of ugly plays from the game for obvious reasons. But at the same time I think the discussion lies in how we do that."
Frazier wasn't sure if Peterson was aware of the proposal but was certain he knew which way the MVP would lean.
"He would not be in favor," Frazier said.
"Those guys have to be instinctive. They have to be allowed to do what they do."


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- Series 11. Data From the National Health Examination Survey, the National Health and Nutrition Examination Surveys, and the Hispanic Health and Nutrition Examination Survey
- Series 12. Data From the Institutionalized Populations Surveys
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- Series 22. Data From the National Mortality and Mortality Natality Surveys
- Series 23. Data From the National Survey of Family Growth
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Paper copies may be available at a Government Depository or a health sciences library near you. Highest numbers are most recent publications.
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A Birthday cake was needed! I just recived a new issue of Good Food direct from England......inside was a mini-edition of Olive Magazine. It was filled with some interesting recipes, including a lovely Chocolate Swirled Cheesecake from non other than Chef Gordon Ramsay. The bête noire, who was getting some press 'down under'!
The ingredients included Double Cream(hard to come by in the US), Creme Fraiche, and some high cocoa chocolate (brought over from France)! I set off to Lexington to Whole Foods.....just to see! I knew they carried Creme Fraiche! What a surprise to find Double Cream, and boy-oh-boy.....this was thick and delicious! The recipe called for Digestive Biscuits, but I had Chocolate Graham crackers on hand and thought I would substitute!
Now you see why I call this the United Nations Cheesecake! A real combo of ingredients. The beauty of this recipe is it is non-bake.......so no cracking and fiddly baking. I combined and swirled and then we celebrated a birthday and fought over the last crumb. Creamy, smooth, a hint of lemon and that wonderful rich chocolate. Even the crust set off the taste of the cheesecake!


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- Do Draenei get sunburn, and if so do they end up looking like Eredar?
- Why do Sindorei --who often have names invoking their love of daylight-- seem to never get tan?
- With all the radiation surrounding Gnomeregan, why don't more of the Gnomes turn into mini-Hulks?
- How can some of the Imperial NPCs --the regular populace-- tell you that they're loyal servants of the Empire without breaking down into laughter?
- In Guild Wars 2, when you aggro an enemy bandit they say "Ha! You're kidding, right?" Why don't they say as they die "I guess you weren't kidding..."
- When I see a quest giver in Neverwinter talk, why is it like watching a dubbed martial arts movie?
- Why do Gnome NPCs say "Daylight's burning!" Shouldn't the Kaldorei say that?
- Given all of the terror that NPC necromancers generate in Age of Conan, why do the inhabitants of the cities never bat an eyelash when a PC necromancer walks by with a bunch of undead trailing in their wake?
- Why are Warlocks a playable class in WoW, anyway? Aren't they supposed to be the bad guys?
- Why is the scenery so great in LOTRO, but the toon graphics so... blah?
- Is "multiplicity" Garrosh's super power? After all, he's in Orgrimmar, he's in Borean Tundra, he's in Nagrand, and oh look he's over at the Argent Tournament Grounds and at Silverpine Forest...
- "Evil will always triumph because Good is dumb," says Dark Helmet. Well.... let me introduce you to Darth Malgus....
- What do jawas/sandpeople look like underneath those robes, anyway? On second thought, maybe I don't want to know; it could be Gargamel and the Smurfs.
- What sort of birth control do they use on those Bioware games? Sure seems like you'd expect a lot more kids around than what you see in game.
- Why is it that the younger a player is, the louder they feel they have to be in BG chat? It's not a perfect correlation, but it sure seems that way at times.
- How can kindergartners sit in "circle time" without any trouble at all, but a bunch of BG players can't stand within a circle in Silvershard Mines?
- Why do the WoW male human toons all look like they've got gas?
Tuesday, October 15, 2013
Some Ponderables
7 comments:
"Why are Warlocks a playable class in WoW, anyway? Aren't they supposed to be the bad guys?ReplyDelete
Do you know, I always wonder why any alliance adventurer wants to be a warlock. But as for the bad guys, we already have a bunch of those: the Horde. I don't know why the Tauren are on that side. We should kick warlocks out of the alliance, and ask the Tauren if they'll join us.Replies
They're bad guys even on the Horde side. You spend a lot of time on the Horde side dealing with the Burning Blade in Durotar and the Barrens in the pre-Cata days --not the same now-- and that all the Warlocks were slain in the Cleft of Shadow prior to Garrosh's last stand is a testament to how much the Horde hate the demons.Delete
As for the Tauren, there's no way they'd join the Alliance after Camp Taurajo.
That old chestnut! In Camp Taurajo, 4 tauren died. None of them killed by alliance adventurers. By contrast, tauren adventurers took part in the assault on Theramore. Nonetheless, I still think Tauren are not naturally horde.Delete
- AnonymousOctober 18, 2013 at 12:31:00 PM EDT
Things I never though of except for the Warlock one. Okay, I'll send my Draenei to a beach vacation in Tanaris and I'll get back to you on that one!ReplyDelete
Are you saying that Darth Malgus is dumb? :PReplyDelete


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The Xbox One Kinect Sensor uses a microphone to hear your voice commands. The key to a great experience is making sure your Kinect sensor can hear you. This article describes how to troubleshoot issues with the Kinect microphone when you try to use voice commands.
Note Xbox One voice commands are not available in all regions. To find out if voice options are available in your country or region, see Xbox One Feature Availability. Check the Premium/Natural Voice (including "Xbox On") feature to see if it's available in your locale.
Consider the following points when you adjust the Kinect microphone or configure your play space:
If you're having trouble within a specific game or app, view the game or app's tutorial or try to calibrate the Kinect sensor from within that game or app.
If you find you're still having problems with a specific game, go to the Xbox One Games forums for tips and help from the community.
If you're having trouble getting your Kinect sensor to recognize voice commands, try the following.
Verify that your Kinect sensor is on
Restart your console
Note If the console appears to be frozen, press and hold the Xbox button on the console for 10 seconds, until the console turns off. After the console shuts down, touch the Xbox button on the console again to restart.
Calibrate the Kinect microphone
Before you start the procedure:
During the audio check, the sensor checks the background noise in your play space. If there isn't too much background noise, the system automatically goes to the next step. If there is too much background noise, minimize the noise and then run the audio check again.
During the audio check, the sensor plays several tones to determine speaker placement, and the system displays a screen when it calibrates the microphone. This may take a few seconds.
If you continue to experience the problem, request a Kinect sensor repair at Device service and repair. Console registration is required to replace the sensor.
Thanks for your feedback!
Provide feedback for this topic
Would you like to contact Support regarding the "Troubleshoot issues with Kinect voice commands on Xbox One" issue you were reading about or a different issue?


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Create New Account
By creating an account you will be able to shop faster, be up to date on an order's status, and keep track of the orders you have previously made. Registered customers are also automatically enrolled in our VIP program letting you earn points for every purchase redeemable for gift cards and other perks.
Note: Creating an account is optional and you can checkout as a guest. Just add items to your cart, choose checkout, then checkout as a guest.


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Author: Marie Yates Taylor
I'm living on the Texas Gulf Coast, where weather quickly changes. I post weather, disaster-related infos I receive, and weather history. And ... much more. Add your stories, and post along with me about your area too. Surviving is all about the public and informed preparedness. View all posts by Marie Yates Taylor


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Back to Normal Mode! The team of HyipNews is back after Christmas and New Year holidays! The end of festive period means complete return to the normal work mode. The editorial, monitoring and support department are doing their best to provide all customers with timely and professional news, hyip investment programs updates and high-quality support on any possible issues that may arise.
Happy Thanksgiving Day, dear readers! Despite the fact that most major holidays mean breaks and new risks for the HYIP industry, I suppose there is no such thing as bad holidays. And Thanksgiving day is a very special holiday. The team of HyipNews wishes you a wonderful family life and big opportunities in the future as well as as much as possilble profitable and reliable high yield investment experience.
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Data breaches are reported all the time in the news - and often when a breach occurs, fingers are pointed at everyone from hackers, to CSOs, IT and even end users. So in looking at a typical breach, Jackson Shaw, senior director of product management at Quest Software, wanted to break down why the fingers get pointed at these particular people (aside from the hacker, as that's generally an obvious target) and why they should each care about data governance. For simplicity, he's going to break this down into three groups: The end user, the IT department, and the line of business manager, and he'll use the case of financial data.
Related Topics : Transport Layer Security, Encryption, SEO, Switches
Unearth the real story behind five commonly held myths about distributed denial-of-service attacks. ... More >>
When a security breach occurs, IT teams that are armed with incident response checklists will be better prepared to execute a fast and effective response. ... More >>
Five common failures companies make when preparing for, and responding to, a data breach, as well as guidance for companies on how they can tackle these issues. ... More >>


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Hello Kitty PS Vita Game Holds Open Casting Call
posted on by Jennifer Sherman
The makers of the Hello Kitty to Issho! Block Crash V PS Vita puzzle game are holding an open casting call from Friday until December 9. Two candidates will be selected to voice the new "Kittyler" characters Charlotte, a confident princess, and Kanon, a gentle girl.
Applicants must be at least 20 years old, but the casting call is open to men as well as women. The game's creators will make the first selection based on candidates' resumés and voice samples. Applicants who pass through to the second phase will submit recordings of the characters' lines.
Japanese game development company Dorasu is collaborating with Sanrio's popular Hello Kitty to Issho! (With Hello Kitty!) project to release the Hello Kitty to Issho! Block Crash V. Dorasu released Hello Kitty to Issho! Block Crash 123!!, the first Hello Kitty to Issho! puzzle game, in July 2010 on the PSP. The new PS Vita game's release date has not been set.
Di Gi Charat illustrator Koge Donbo created one of the more recent "Kittyler" characters named Moe Kirishima. Other artists who drew characters for the project include Okama, Kouhaku Kuroboshi, POP, Akihiko Koizumi, Shimeko, Keito Koume, Chinatsu Kurahana, Eiji Komatsu, KEI, Poyoyon Rock, Milk Morinaga, Aoi Nishimata, Nisieda, Carnelian, and redjuice. The new characters' artists have not been announced.
[Via Hachima Kikō]
this article has been modified since it was originally posted; see change history


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Hospira ends shutdown after corrective steps at NC plant
The production plant in Clayton, North Carolina was cited in a US Food and Drug Administration (FDA) warning letter in 2010 and since then Hospira has worked to improve quality. However, in March there was a blip which affected some of Hospira's contract manufacturing clients.
"During the month of March, we encountered some manufacturing issues at the plant and elected to shut down production to investigate. This has...impacted certain contract manufacturing customers," Michael Ball, CEO of Hospira, told investors this week.
The shutdown also created a temporary shortage of propofol. Last week Hospira completed its efforts to fix the problem and has restarted manufacture. A staged release of quarantined products is expected to begin in the coming weeks.
Efforts to fix the Clayton site are part of a broad quality push at Hospira as it looks to recover from multiple regulatory enforcement actions in recent years. A current focus on the work is the Rocky Mount production plant in North Carolina which featured in the same warning as Clayton.
"We have placed over 100 full-time employees at the plant since the beginning of the year. This is in addition to the more than 150 consultants we already have working on the plant floor with our employees and assisting us with our remediation efforts," Ball said.
Investments in quality, plus relating factors like inventory losses, dragged gross margins down by five percentage points year-on-year. Gross margins are expected to drop further in the back half of 2012 as Hospira contends with scheduled - but extended - maintenance shutdowns.


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I was so very happy to take photographs for the LifeWorks Home Improvement Challenge. My friend Christen Ales was one of the interior designers. She partnered with volunteers from Dell for this amazing opportunity to do something very special for a very deserving family. Christen and the Dell team worked so hard to create a beautiful living space for a young woman and her daughter. Most of the items were either donated, found, or made by hand (check out the awesome headboard Christen made!)
I am so proud to have been there for the reveal. Just seeing the expression on the young woman's face was priceless. It is inspiring to know that this makeover will give this family a safe beautiful space of their own to help them create positive changes in their lives. For more information on LifeWorks and what they do, please visit their website. Better yet, donate or volunteer your time to help provide at-risk youth and families in crisis with the resources they deserve to succeed.