Nature Methods Application Notes e-UPDATE: 12 November 2013

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12 NOVEMBER 2013  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 High-content analysis of three-dimensional tumor spheroids: investigating signaling pathways using small hairpin RNA www.3dbiomatrix.com > www.ttplabtech.com > http://www.gnf.org > In the past, creating three-dimensional (3D) tumor spheroids that were suitable for high-throughput screening (HTS) was a difficult and often expensive process. We describe how to couple easy, controllable 3D spheroid formation in Perfecta3D® Hanging Drop Plates with the acumen® eX3 high-content imager for rapid, multicolor, whole-well quantification. This process provides researchers with a highly efficient method to achieve physiologically relevant tumor models in an HTS format. PDF

		Automation of ChIP-Seq Library Preparation for Next Generation Sequencing on the epMotion®5075 TMX http://www.eppendorfna.org > ChIP-Seq library preparation can be a challengingprocedure to automate because of its low ChIP DNAinput. This Application Note describes the successfulautomation of Illumina™ChIP-Seq library preparation onthe Eppendorf®epMotion 5075 TMX, using the KAPAHigh-Throughput Library Preparation Kit from KAPABiosystems®. Size-selected libraries were prepared fromas little as 1 ng of fragmented ChIP DNA. To obtain the recommended 100 ng of library material forsequencing on the Illumina Genome AnalyzerIIx, only 18amplification cycles were needed for 1 ng of input DNA,or 9 cycles when starting library construction with 10 ngof ChIP DNA. PDF

		A Novel Molecular Approach for Measuring Viral Load www.abionline.com > Advances in molecular techniques have initiated a revolution in the field of infectious diseases. Molecular assays are now routinely used to measure viral load. Many of these assays require a critical nucleic acid extraction step, followed by quantitation of recovered DNA. Extraction methods, however, inherently suffer from inefficiencies in individual steps and loss of nucleic acid during the extraction process. While there are many different methods and technologies available for nucleic acid extractions, none of these include appropriate controls to monitor extraction efficiency and loss of DNA. To ensure reliable and reproducible results, there is a need for appropriate extraction controls to validate nucleic acid extraction methods, and standards for nucleic acid quantitation. PDF

		Trypsin/Lys-C protease mix for enhanced protein mass spectrometry analysis www.promega.com > Traditionally, trypsin and Lys-C proteases are used in combination to digest proteolytically resistant proteins. Here we describe use of Trypsin/Lys-C Mix for general digestion needs. Working simultaneously under conventional non-denaturing trypsin digestion conditions, Trypsin and Lys-C enhance proteolysis and provide multiple positive effects on protein mass spectrometry analysis including increased number of identified peptides and proteins, higher analytical reproducibility and more accurate protein quantitation. Effectively, by supplementing trypsin with Lys-C, we create improved trypsin. PDF

		Optimized method for rapid protein electroblotting www.thermoscientific.com > Protein electroblotting is a common method for transferring proteins out of a polyacrylamide gel onto a membrane for subsequent western blotting. With a high-current power supply, such as that found in the Thermo Scientific™ Pierce™ G2 Fast Blotter, and a high–ionic strength transfer buffer, protein transfer from nitrocellulose or polyvinylidene difluoride (PVDF) membranes can be achieved in 5–10 m, even with proteins as large as 300 kDa. PDF

		EpiGnome™ Methyl-Seq Kit: a novel post–bisulfite conversion library prep method for methylation analysis www.epibio.com > Epigenomics is increasingly becoming an important field of research, and the ability to detect and quantify DNA methylation accurately is now critical for numerous fields of study, including disease biology and gene expression. The differential reactivities of methylated and nonmethylated cytosines in DNA with sodium bisulfite forms the basis for their identification in the genome by sequencing. Current whole-genome bisulfite sequencing methods require substantial amounts of starting DNA to compensate for the loss due to bisulfite-mediated DNA degradation. To address issues with sample preparation, we have developed the EpiGnome™ Methyl-Seq Kit, which utilizes a novel pre-library bisulfite conversion scheme to prepare whole-genome bisulfite sequencing libraries with no sample loss and with only 50 ng of input genomic DNA. PDF

		An Integrated Solution to Simplify Library Preparation and Multiplexing for NimbleGen Sequence Capture http://www.biooscientific.org > Targeted sequencing is an important tool in analyzing disease or exome mutations. In this study, we describe how Bioo Scientific's NEXTflex™ DNA Pre-Capture Combo Library construction kits were used in conjunction with NimbleGen Sequence Capture technology to obtain high coverage comparative genomic data from a panel of human HapMap DNA samples. We identified 96-98% of known HapMap SNPs in the NimbleGen SeqCap EZ Exome v3.0 (64 Mb) and SeqCap EZ Design — Comprehensive Cancer Design (3.9 Mb) probes with a high percentage of reads mapping to the targeted regions. This study illustrates both the ability of Bioo Scientific's NEXTflex library construction kit to produce high-quality material for NGS, and the robust performance of the NimbleGen Sequence Capture technology. PDF

 

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