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RapidFinder™ STEC Confirmation Assay (Applied Biosystems™)

The Applied Biosystems™ RapidFinder™ STEC Confirmation Assay is part of a complete, validated screening and confirmation workflow enabling TaqMan™ real-time PCR detection of the Shiga toxin-producing E. coli (STEC) O157:H7 and Big 6 non‐O157:H7 serogroup (O26, O45, O103, O111, O121, and O145) in a variety of beef products. Samples assessed as positive using the RapidFinder STEC Screening Assay are further tested using this RapidFinder STEC Confirmation Assay. All reactions include an internal positive control (IPC) and ROX™ passive reference.

The RapidFinder STEC Detection Workflow provides:
• Greater confidence in results—part of a validated workflow for detection of as little as 1-5 CFU of pathogenic STEC in your sample
• Simplified workflow—simultaneous detection of E. coli O157:H7 and the Big 6 non-O157:H7 strains in 375 g beef samples with a closed-tube, single-step lyophilized detection format that fits within your existing testing program
• Reduced chance of false results—through use of high-quality DNA preparation, proprietary assay design, and internal positive control
• Same day detection of pathogenic STECs and clearance of STEC-negative beef samples in as little as 12 hours, supporting early product release
• Minimized costs and laboratory space with the use a lower volume of a nonproprietary single-step enrichment medium, eliminating the need to purchase proprietary media
• Compatibility with RapidFinder™ Express software v.1.2 and later—enables easy, automated presence/absence detection and interpretation of results

Studies have shown that the RapidFinder STEC assays have a high level of sensitivity and specificity for E. coli O157:H7 and the Big 6 non-O157 STEC strains*. They detected all inclusion strains and showed no cross-reactivity to any of the exclusion strains tested. The stx assays detected all known variants of stx1 and stx2, including stx2f and stx2g. The optimized workflow co-developed with USDA-ARS showed equivalent detection to the MLG reference method, and the internal positive control in both assays significantly reduces the risk of false-negatives.

Applications
The RapidFinder STEC Detection Workflow is intended for use by microbiological analysts who need to test for STEC in beef samples. Combined with the Applied Biosystems 7500 Fast RT-PCR System, RapidFinder STEC screening and confirmation assays provide a comprehensive solution for rapid detection, identification, and confirmation of Big 6 STECs and E. coli 0157:H7 to meet importation and exportation requirements for the beef industry. Specific workflows are detailed in the figures below and include both automated and spin column-based DNA isolation and real-time PCR detection of E. coli O157:H7 and Big 6 non-O157 strains in beef samples.

RapidFinder Express Software
When integrated into the RapidFinder STEC Detection Workflow, RapidFinder Express Software automates data interpretation and generates user instructions on how to proceed with a sample. Data displays are available for novice and advanced users, showing simple presence/absence calls or raw data files using 7500 Fast System SDS Software.

RapidFinder STEC Screening and Confirmation Assays meet USDA FSIS guidance
The RapidFinder STEC Detection Workflow, a two-stage real-time PCR method, is designed to accurately detect, identify, and confirm E. coli O157:H7 and the Big 6 serogroups of non-O157 Shiga toxin-producing E. coli defined by the USDA Food Safety and Inspection Service (FSIS) as adulterants in the American beef industry.

Learn more about the RapidFinder STEC Detection Workflow >

*Swimley, Michelle S. Design and Evaluation of Two-Stage Multiplex Real-Time PCR Method for Detecting O157:H7 and non-O157 STEC Strains from Beef Samples. IAFP 2015.

MAGnify™ Chromatin Immunoprecipitation System (Applied Biosystems™)

The MAGnify™ Chromatin Immunoprecipitation System provides a streamlined, optimized assay for the enrichment of chromatin/protein complexes and DNA recovery using magnetic bead capture technology. The isolated DNA is ready for downstream analysis by methods such as PCR- or qPCR-based assays, or massive parallel DNA sequencing.

ChIP
Chromatin immunoprecipitation (ChIP) is a powerful technique for studying the association of certain proteins with specific regions of the genome. These sequence-specific DNA-binding proteins are believed to play a role in such cellular processes as DNA replication, recombination, repair, and segregation; chromosomal stability; cell-cycle progression; and epigenetic silencing. In a standard ChIP assay, a cell is fixed via formaldehyde treatment and the chromatin is sheared and immunoprecipitated via a highly specific antibody. The researcher then analyzes the DNA to identify the genomic regions where the chromatin-associated proteins bind to the chromatin in vivo. This kit enables researchers to start with lower sample amounts than traditional ChIP workflows, thereby preserving precious samples, and the protocol can be completed in a single day, compared with 2–3 days for a traditional ChIP assay. The kit can be used with our suite of ChIP-validated antibodies, and is also complementary with MethylCode™ and NCode™ products for downstream epigenetics research.

Using the MAGnify™ system
When using the MAGnify™ system, you treat cells or tissue with formaldehyde to generate protein-protein and protein-DNA crosslinks between molecules in close proximity within the chromatin complex. The cells are then lysed, and the chromatin is released from the nuclei and sheared by sonication to reduce the average DNA fragment size to 200–500 bp for analysis by quantitative real-time PCR (qPCR) or 100–300 bp for analysis by massive parallel DNA sequencing. You then immunoprecipitate and isolate the crosslinked protein of interest using a specific ChIP-qualified antibody conjugated to Dynabeads® Protein A/G. The formaldehyde crosslinking is reversed by heat treatment, and the DNA associated with that protein is purified. The DNA is now ready for downstream analyses such as end-point PCR or quantitative PCR (qPCR), genome-wide analyses using promoter-tiling arrays, or next-generation sequencing. In PCR/qPCR analysis, primers are designed to span the desired DNA sequence of interest, and the data demonstrates whether the specific protein of interest is associated in vivo with that DNA region.

North2South™ Chemiluminescent Substrate for HRP (Thermo Scientific™)

The Thermo Scientific North2South Chemiluminescent Substrate is used for detecting nucleic acids in northern and Southern blot applications. This robust system uses an enhanced luminol substrate for horseradish peroxidase (HRP) that greatly reduces processing and film exposure time. Post-hybridization processing has been reduced from the standard 2.5 hours to 1 hour. Film exposure times range from 30 seconds to 10 minutes with the substrate emitting light with relatively constant intensity for up to 6 hours, allowing for multiple exposures.

Related Products
North2South™ Complete Biotin Random Prime Labeling and Detection Kit
North2South™ Chemiluminescent Hybridization and Detection Kit
North2South™ Hybridization Buffer
North2South™ Hybridization Stringency Wash Buffer (2X)

MEGAscript™ T3 Transcription Kit (Invitrogen™)

Novel transcription reaction conditions and Ambion's patented, high-yield technology allow the synthesis of 10–50 times the amount of RNA produced by conventional transcription reactions. Each Ambion® kit includes sufficient reagents for 40 reactions.

• Exclusive, ultrahigh yield technology is fast: 2 hour reaction time
• Amplifies aRNA for gene array analysis and other applications
• Efficiently incorporates many modified nucleotides

A typical 20 µl MEGAscript® reaction with 1 µg of the pTRI-Xef-1alpha control template will yield over 100 µg of transcript. The versatility of the MEGAscript® kit allows for manipulation of the reactions to include specialized reagents such as modified nucleotides, cap analog, or additional polymerase.

Accessory Products:
MessageAmp™ aRNA Amplification Kits are powered by MEGAscript® technology and have been developed especially for the amplification of cRNA prior to array analysis. To purify in vitro transcription products from free nucleotides, buffer components, and enzymes, Ambion recommends using the MEGAclear™ Kit (SKU#AM1908) and the MEGAclear™-96 Kit (SKU#AM1909).

mMESSAGE mMACHINE™ SP6 Transcription Kit (Invitrogen™)

mMESSAGE mMACHINE® kits are designed for the in vitro synthesis of large amounts of capped RNA. Capped RNA mimics most eukaryotic mRNAs found in vivo, because it has a 7-methyl guanosine cap structure at the 5' end. mMESSAGE mMACHINE® kit reactions include cap analog [m7G(5')ppp(5')G] in an ultra high-yield transcription reaction. The cap analog is incorporated only as the first or 5' terminal G of the transcript because its structure precludes its incorporation at any other position in the RNA molecule.

How mMESSAGE mMACHINE® kits work
mMESSAGE mMACHINE® kits have a simplified reaction format in which all four ribonucleotides and cap analog are mixed in a single solution. The cap analog:GTP ratio of this solution is 4:1, which is optimal for maximizing both RNA yield and the proportion of capped transcripts. mMESSAGE mMACHINE® kits are ideal for the routine synthesis of capped RNAs for oocyte microinjection, in vitro translation, transfection, and other applications. The high yields are achieved by optimizing reaction conditions for RNA synthesis in the presence of high nucleotide concentrations. In addition, the RNA synthesized is protected from degradation by any contaminating ribonucleases that may be present with RNase inhibitor—a component of the Enzyme Mix.

Using mMESSAGE mMACHINE® Kits
In a 20 µL reaction during a 2 hour incubation, mMESSAGE mMACHINE® High Yield Capped RNA Transcription will yield large mass amounts of capped RNA. Up to 10–50 times the yield obtained with conventional in vitro transcription reactions. The ratio of cap analog to GTP has been optimized to allow the best compromise between yield (15–35 µg) and proportion of transcripts that are capped (80%). mMESSAGE mMACHINE® Kits also contain a LiCl precipitation solution that is efficient for separating proteins and unincorporated nucleotides (including free cap analog) from the capped RNA, allowing an increased efficiency of translation. mMESSAGE mMACHINE® kits are only optimized for use with the polymerases included in the kit. Using a different polymerase may result in low yields. The mMESSAGE mMACHINE® kits contain all the buffers and reagents necessary for 25 transcription reactions. Using the control template supplied with the kits (Xenopus elongation factor 1α, pTRI Xef), each mMESSAGE mMACHINE® reaction will yield approximately 20–30 µg of RNA using T3 or T7 RNA polymerase, or about 15–25 µg RNA using SP6 RNA polymerase.

mMESSAGE mMACHINE™ T7 Transcription Kit (Invitrogen™)

mMESSAGE mMACHINE® kits are designed for the in vitro synthesis of large amounts of capped RNA. Capped RNA mimics most eukaryotic mRNAs found in vivo, because it has a 7-methyl guanosine cap structure at the 5' end. mMESSAGE mMACHINE® kit reactions include cap analog [m7G(5')ppp(5')G] in an ultra high-yield transcription reaction. The cap analog is incorporated only as the first or 5' terminal G of the transcript because its structure precludes its incorporation at any other position in the RNA molecule.

How mMESSAGE mMACHINE® kits work
mMESSAGE mMACHINE® kits have a simplified reaction format in which all four ribonucleotides and cap analog are mixed in a single solution. The cap analog:GTP ratio of this solution is 4:1, which is optimal for maximizing both RNA yield and the proportion of capped transcripts. mMESSAGE mMACHINE® kits are ideal for the routine synthesis of capped RNAs for oocyte microinjection, in vitro translation, transfection, and other applications. The high yields are achieved by optimizing reaction conditions for RNA synthesis in the presence of high nucleotide concentrations. In addition, the RNA synthesized is protected from degradation by any contaminating ribonucleases that may be present with RNase inhibitor—a component of the Enzyme Mix.

Using mMESSAGE mMACHINE® Kits
In a 20 µL reaction during a 2 hour incubation, mMESSAGE mMACHINE® High Yield Capped RNA Transcription will yield large mass amounts of capped RNA. Up to 10–50 times the yield obtained with conventional in vitro transcription reactions. The ratio of cap analog to GTP has been optimized to allow the best compromise between yield (15–35 µg) and proportion of transcripts that are capped (80%). mMESSAGE mMACHINE® Kits also contain a LiCl precipitation solution that is efficient for separating proteins and unincorporated nucleotides (including free cap analog) from the capped RNA, allowing an increased efficiency of translation. mMESSAGE mMACHINE® kits are only optimized for use with the polymerases included in the kit. Using a different polymerase may result in low yields. The mMESSAGE mMACHINE® kits contain all the buffers and reagents necessary for 25 transcription reactions. Using the control template supplied with the kits (Xenopus elongation factor 1α, pTRI Xef), each mMESSAGE mMACHINE® reaction will yield approximately 20–30 µg of RNA using T3 or T7 RNA polymerase, or about 15–25 µg RNA using SP6 RNA polymerase.

FISH Tag™ DNA Green Kit, with Alexa Fluor™ 488 dye (Invitrogen™)

FISH Tag™ DNA Kits provide a complete workflow solution for fluorescence in situ hybridization (FISH) applications. Each kit provides all of the reagents needed for probe synthesis, labeling, and purification, as well as for imaging the labeled specimen. Choose from FISH Tag™ DNA Kits in three Alexa Fluor® colors or get the FISH Tag™ DNA Multicolor Kit, which contains four spectrally distinct dyes to allow simultaneous localization of multiple sequence-specific DNA targets in chromosomal spreads and for in situ analysis of mitochondrial DNA localization and content.

FISH Tag™ Kit Specifications:
• Dye (Ex/Em): Alexa Fluor® 488 (492/520 nm)
• Comprehensive kit includes 16 reagents
• Comes with detailed protocol to help ensure your success


Complete Workflow Solution for FISH
FISH Tag™ Kits employ a two-step labeling technology—nick translation to enzymatically incorporate an amine-modified nucleotide (aminoallyl dUTP) followed by chemical labeling with Alexa Fluor® dyes. PureLink® nucleic acid purification technology is used for fast and efficient purification of the labeled probe, and SlowFade® Gold antifade reagent is added to preserve the fluorescent signal during imaging.

More Options for Nucleic Acid Labeling
To review various options for nucleic acid labeling (including DNA and RNA FISH), review Labeling Oligonucleotides and Nucleic Acids—Section 8.2 in the Molecular Probes® Handbook.

For Research Use Only. Not intended for human or animal therapeutic or diagnostic use.

MethylCode™ Bisulfite Conversion Kit (Applied Biosystems™)

The MethylCode™ Bisulfite Conversion Kit is a fast and easy way to convert unmethylated cytosines from a DNA sample into uracils while methylated cytosines remain unchanged.

The modified DNA is ideal for PCR amplification for downstream analyses, including restriction endonuclease digestion, sequencing, and microarrays. With the MethylCode™ Bisulfite Conversion Kit you can expect:

–Simple, rapid cytosine methylation detection

–Consistency across most experimental conditions
–Streamlined conversion of unmethylated cytosines into uracil, thanks to a coupled DNA denaturation/bisulfite treatment step
–Ultra-pure DNA ideal for subsequent molecular-based analyses



Kit specifications:

–Protocol: Rapid heat denaturation of DNA followed by sodium bisulfite modification with spin column purification

–Sample sources: Plasmid, genomic, and endonuclease digested DNAs
–Input DNA: 500 pg-2 µg DNA per treatment, with 200-500 ng being optimal
–Recovery volume: Purified, bisulfite-treated DNA is recovered in 10 µl elution buffer



Contents and storage:
The MethylCode™ Bisulfite Conversion Kit contains all the reagents necessary to convert your DNA for methylation studies. The kit includes CT Conversion Reagent, Dilution Buffer, Resuspension buffer, Binding Buffer, Wash Buffer, Desulphonation Buffer, Elution Buffer, Spin Columns, Collection Tubes.

Store at room temperature for up to 24 months.

MAXIscript™ SP6 Transcription Kit (Invitrogen™)

The Ambion® MAXIscript® SP6 In Vitro Transcription Kit synthesizes RNA probes in just 10 minutes for use in ribonuclease protection assays, in situ hybridization, and blot hybridizations. Each kit contains sufficient reagents for 100 reactions.

Features:

• High yields of high specific activity, full-length RNA probes in just 10 minutes
• Ideal probe synthesis kit for nuclease protection assays, northern and Southern blots, and in situ hybridization
• Includes TURBO DNase™
• All kits contain quality-tested DNase/RNase-free reagents

The MAXIscript® In Vitro Transcription Kit synthesizes RNA probes with specific activities reaching 1 × 109 cpm/µg in just 10 minutes. MAXIscript® Kits are very efficient at synthesizing full-length probes, even at limiting nucleotide concentrations, making them the perfect companion to ribonuclease protection assays. MAXIscript® Kits are also an excellent choice for the synthesis of nonisotopically labeled probes. Labeling can be accomplished post-transcriptionally by using Ambion's BrightStar® Psoralen-Biotin Labeling Kit, or during transcription by incorporating modified nucleotides into the reaction. MAXIscript® Kits can also be used for the synthesis of modest amounts of unlabeled RNA (2–6 µg/20 µl reaction) of up to 2 kb.

RapidFinder™ Equine ID Kit

The RapidFinder™ Equine ID Kit enables the simple and reliable detection of horse meat in food and feed samples. The method works by detection of horse (Equus caballus) DNA by real-time PCR. Unlike ELISA based methods, the RapidFinder™ Equine ID Kit detects both raw and processed horse meat and provides sensitivity down to 0.01% horse meat DNA when used with the GMO Extraction Kit. A positive control is included in the kit (0.1% equine DNA) that can be used as a reference to set the threshold of detection of the assay.

• Simple—real-time PCR based assay, no electrophoresis needed
• Flexible—equine DNA detected in any food or feed sample, both raw and processed
• Reliable—internal positive control (IPC) provides process control check for PCR inhibitors
• Fast—from sample to result on the same day

How the RapidFinder™ Equine Kit Works
Equine DNA detection is accomplished by real-time PCR using the supplied primer/probe set specific for a mitochondrial DNA sequence that is unique to Equus caballus. The target sequence, detected in the FAM™ channel, is not only specific for horse, but also stable. Included in each reaction mix is an internal positive control detected in the VIC® channel, used to verify the absence of PCR inhibitors.

By performing a separate reaction with the included positive control, users are able to verify the system and assay are working correctly. More importantly, it enables easy data interpretation. The positive control is provided as 0.1% horse DNA in a background of beef DNA, and by comparing sample results to control results, a threshold of detection is provided.

The assay is compatible with Applied Biosystems® StepOne™, StepOnePlus™, 7500, 7500 Fast, and 7300 real-time PCR systems.

Sample Preparation
DNA sample preparation for the RapidFinder™ Equine ID Kit workflow is accomplished with the easy-to-use GMO Extraction Kit. Using 10 g of sample rather than the smaller sample sizes accommodated by most other DNA isolation procedures minimizes sampling errors. The GMO Extraction Kit is compatible with raw and processed meat, as well as composite food samples.

Quantification
If a positive result is obtained with the RapidFinder™ Equine ID Kit, you can quantify the amount of equine DNA in the sample using the RapidFinder™ Quant Equine Set.

ViewRNA™ Tissue Assay Blue Module (Invitrogen™)

This VewRNA Tissue Assay Blue Module is designed for use with the ViewRNA Tissue Assay Core Kit. The Core Kit, when combined with a ViewRNA Tissue probe set (purchased separately), enables the detection of a single mRNA target in tissue sections using the Fast Red substrate. The ViewRNA Tissue Assay Blue Module enables the detection of a second mRNA target using the Fast Blue substrate and must be used in combination with the Core Kit and two probes sets in order to run a 2-plex assay. Each kit contains sufficient reagents to process a minimum of six slides at a time. ViewRNA TYPE 1 probe sets are for use with the Fast Red substrate (Core Kit), while ViewRNA TYPE 6 probe sets are for use with the Fast Blue substrate (Blue Module).

Learn more about and order ViewRNA Tissue probe sets ›

ViewRNA Tissue assays are RNA in situ hybridization (ISH) assays for the reliable detection of one or two mRNA targets within tissue sections. Visualize any gene in any tissue with single-copy sensitivity and no radioactivity. And in case your needs are not met by one of our 6500 catalog probe sets, we have decades of bioinformatics expertise and will design custom probe sets to any mRNA target of interest, free of charge.

With ViewRNA Tissue assays, you can achieve:
• Robust, single mRNA molecule detection in tissue sections
• Accuracy and specificity inherent to branched DNA amplification technologies
• Simultaneous detection of two RNA targets

The ViewRNA Tissue Assay is compatible with formalin-fixed, paraffin-embedded (FFPE) tissue or OCT (optimal cutting temperature) embedded frozen tissue sections. Whereas traditional ISH technologies often amplify both signal and background, ViewRNA assays use branched DNA (bDNA) technology to amplify target-specific signal and achieve superior signal-to-noise ratios.

mMESSAGE mMACHINE™ T7 ULTRA Transcription Kit (Invitrogen™)

The mMESSAGE mMACHINE® T7 Ultra Kit combines a new cap analog, anti-reverse cap analog (ARCA), with a patented high-yield transcription technology to generate RNA transcripts that produce higher protein yields compared to other transcripts upon translation. Advantages of the mMESSAGE mMACHINE® T7 Ultra Kit:

• Synthesize more protein from in vitro-transcribed RNA
• Express RNA transcripts more efficiently both in vitro and in vivo
• Stabilize transcripts in vivo with included reagents for poly(A) tailing

ARCA-capped RNA is translationally more active
A base modification in ARCA results in its incorporation in the functional, translatable orientation only; traditional cap analog can be incorporated in both functional and nonfunctional orientations. As a result, incorporating ARCA into transcription reactions yields capped RNAs that are 100% translatable. This is further enhanced by the inclusion of poly(A) tails in mMESSAGE mMACHINE® T7 Ultra transcripts. Experiments comparing ARCA and ARCA/poly(A)-tailed transcripts to cap analog and cap analog/poly(A)-tailed transcripts indicate higher levels of protein synthesis with ARCA capped RNA .

What Is ARCA?
ARCA is a modified cap analog in which the 3' OH group (closer to m7G) is replaced with OCH3 (see schematic). Because of this substitution, the RNA polymerase can only initiate transcription with the remaining hydroxyl group, thus forcing ARCA incorporation in the forward orientation. As a result, 100% of the transcripts synthesized with ARCA at the 5' end are translatable, leading to a strong stimulatory effect on translation.

Proper capping of in vitro transcribed RNA
Proper capping of RNA promotes correct initiation of protein synthesis, as well as stability and processing of mRNA in vivo. Uncapped RNA is rapidly degraded by cellular RNases after microinjection or transfection into cells. Capped RNA is also typically translated more efficiently in in vitro translation systems, generating RNAs with cap analog incorporated only in the functional orientation. Therefore, substitution of traditional cap analog with ARCA results in the synthesis of capped RNAs that are 100% translatable.

Fast Red Substrate (20 mL Naphthol Buffer + 4 Fast Red tablets) (Invitrogen™)

Fast Red Substrate (20 mL Naphthol Buffer + 4 Fast Red tablets) is an individual component of the ViewRNA™ Chromogenic Signal Amplification Kit, 24 assays. It is a red substrate used for the detection of alkaline phosphatase activity.

mirVana™ miRNA Detection Kit (Invitrogen™)

The Ambion® mirVana™ miRNA Detection Kit provides a fast and sensitive method for detecting small RNAs. The assay is 100–500 times more sensitive than Northern analysis, it is able to detect as little as 10 attomoles (10-17 mol) of target RNA. Each kit includes sufficient reagents for 100 reactions.

• Sensitive—detect miRNA or siRNA in as little as 50 ng total RNA
• Specific—extremely low backgrounds
• Simple and fast—single-tube procedure eliminates the need to transfer to membrane for hybridization
• Multiple target detection—detect multiple small RNAs and mRNAs in the same sample

Rapid Procedure Based on Solution Hybridization
The mirVana™ miRNA Detection Kit is based on a simple solution hybridization principle where the RNA sample is simply mixed with radiolabeled RNA probe(s) and hybridized. Unhybridized RNA and excess probe are removed by a digestion step and the hybridized, protected RNA fragments are then recovered using a patented single-step technology for simultaneous ribonuclease inactivation and nucleic acid precipitation. RNA samples are then resuspended and analyzed on a denaturing polyacrylamide gel.

Simultaneous Detection of siRNA Expression and Target Gene Knockdown
Because multiple RNA species of the same or differing size can be detected in the same sample, the mirVana™ miRNA Detection Kit is ideal for correlating siRNA expression levels with target mRNA knockdown. An example of this application is shown in the accompanying data, which demonstrates that GAPDH mRNA levels were reduced in cells expressing a GAPDH siRNA, but not in cells expressing a control siRNA.

Accessory Products:
The mirVana™ miRNA Probe Construction Kit (SKU#AM1550) is available for the preparation and labeling of probes by in vitro transcription and the mirVana™ Probe & Marker Kit is available for 5' end labeling of oligonucleotide probes and the included Decade Markers (SKU#AM1554).