Nature Methods Application Notes e-UPDATE: 13 March 2012

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13 MARCH 2012  Application Notes bring you the latest information about innovative tools and technologies and their applications in the lab. We hope that you will find this service useful and informative and encourage you to sign up for future Updates to ensure that you never miss one!

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FEATURED APPLICATION NOTE A rapid, directional RNA-seq library preparation workflow for Illumina® sequencing www.epibio.com > Most current RNA-seq library preparation methods are time-consuming, multistep processes. We describe a workflow that includes the Ribo-Zero™ and ScriptSeq™ v2 Kits that enables researchers to go from total RNA to cluster-ready RNA-seq libraries in less than 1 d. The RNA-seq libraries produced are virtually free of contaminating ribosomal RNA (rRNA) and provide for directional paired-end and multiplex sequencing on Illumina® sequencing platforms. PDF

		Expresso® CMV system: effortless mammalian expression cloning www.lucigen.com > The Expresso® systems dramatically increase the speed and efficiency of target gene cloning and protein expression. With Expressioneering™, PCR products are cloned instantly and directionally into pre-processed mammalian expression vectors without sample cleanup or enzyme treatment. PDF

		Novel Approach in Identification of Substrates for Ligases: Protein Microarrays www.lifesensors.com > Dedicated to providing pioneering reagents as well as proteomic service solutions to all questions ubiquitin related, LifeSensors scientists can turn questions into answers. Perhaps no question is more commonly asked of the ubiquitome than “what are the substrates of my E3?”. A combination of protein microarray expertise and novel ubiquitin research reagents makes LifeSensors uniquely qualified to get you those answers. PDF

		Duolink-“In-cell Co-IP” for visualization of protein interactions in situ www.olink.com > Duolink® offers a user-friendly solution for studying protein-protein interactions. Duolink utilizes a pair of antibodies that are capable of quantitatively reporting even weak and transient protein-protein interactions in natively expressing cells either as countable, bright fluorescent spots for a standard fluorescence microscope or via chromogenic detection for brightfield microscopes. It offers all the benefits of traditional coimmunoprecipitation (Co-IP) and western blotting but with better quantitative precision and more information about cell-to-cell variability and target localization. It is also amenable to high throughput in 96 or 384 wells. PDF

		Generation of cell lines using Epstein-Barr Virus (EBV) transformation of small volumes of cryo-preserved whole blood and the use of bench-top flow cytometry to achieve high and reproducible success rates www.hpacultures.org.uk > ECACC has developed a procedure for the generation of lymphoblastoid cell lines (LCLs) by the direct EBV transformation of small amounts (800µl) of cryo-preserved whole blood eliminating any requirement for prior separation of peripheral blood lymphocytes (PBLs). The ability to directly transform frozen whole blood avoids the cost of separating and storing PBLs while retaining the option to make a cell line at any time from all or a subset of the collection. Consistent transformation success rates of over 90% have been achieved using bench-top flow cytometry to track lymphoblastoid proliferation from source blood that has been cryo-preserved for more than four years. PDF

		EndoLISA®: a novel and reliable method for endotoxin detection www.hyglos.de > A new test for the sensitive detection of endotoxin has been developed, based on a lipopolysaccharide-selective, precoated microplate and a factor C–based detection reagent and presented in a complete kit format. The selective capture of lipopolysaccharide (LPS) is achieved using a phage-derived receptor protein exhibiting high affinity and high specificity for the conserved core region of LPS. After binding of sample-LPS to the microplate as the first stage of the assay, the original sample matrix is washed off, thereby eliminating potentially interfering components. In the second stage of the assay, LPS is detected by factor C in a process whereby the principal receptor of the Limulus amoebocyte coagulation cascade reacts with a fluorescence substrate. The new endotoxin test EndoLISA has a detection range from 0.05 EU/ml up to 500 EU/ml. PDF

		Ribo-Zero Gold Kit: improved RNA-seq results after removal of cytoplasmic and mitochondrial ribosomal RNA www.epibio.com > Ribosomal RNA (rRNA) constitutes the majority (>98%) of total RNA preparations. To avoid wasting sequencing reads, it is necessary to remove this abundant RNA before preparing RNA libraries for deep sequencing. We previously developed Ribo-Zero™ technology for cytoplasmic rRNA removal from intact and degraded RNA. Here, we describe the Ribo-Zero Gold Kit: an improvement to the original Ribo-Zero method that allows removal of mitochondrial as well as cytoplasmic rRNA from total RNA preparations. PDF

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