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Current Protocols in Molecular Biology

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https://www.readbyqxmd.com/read/28369679/preparing-viable-single-cells-from-human-tissue-and-tumors-for-cytomic-analysis
#1
Nalin Leelatian, Deon B Doxie, Allison R Greenplate, Justine Sinnaeve, Rebecca A Ihrie, Jonathan M Irish
Mass cytometry is a single-cell biology technique that samples >500 cells per second, measures >35 features per cell, and is sensitive across a dynamic range of >10(4) relative intensity units per feature. This combination of technical assets has powered a series of recent cytomic studies where investigators used mass cytometry to measure protein and phospho-protein expression in millions of cells, characterize rare cell types in healthy and diseased tissues, and reveal novel, unexpected cells. However, these advances largely occurred in studies of blood, lymphoid tissues, and bone marrow, since the cells in these tissues are readily obtained in single-cell suspensions...
April 3, 2017: Current Protocols in Molecular Biology
https://www.readbyqxmd.com/read/28369678/mapping-transposon-insertions-in-bacterial-genomes-by-arbitrarily-primed-pcr
#2
José T Saavedra, Julia A Schwartzman, Michael S Gilmore
Transposons can be used to easily generate and label the location of mutations throughout bacterial and other genomes. Transposon insertion mutants may be screened for a phenotype as individual isolates, or by selection applied to a pool of thousands of mutants. Identifying the location of a transposon insertion is critical for connecting phenotype to the genetic lesion. In this unit, we present an easy and detailed approach for mapping transposon insertion sites using arbitrarily-primed PCR (AP-PCR). Two rounds of PCR are used to (1) amplify DNA spanning the transposon insertion junction, and (2) increase the specific yield of transposon insertion junction fragments for sequence analysis...
April 3, 2017: Current Protocols in Molecular Biology
https://www.readbyqxmd.com/read/28369677/direct-isolation-of-seamless-mutant-bacterial-artificial-chromosomes
#3
George T Lyozin, Yasuhiro Kosaka, Gourab Bhattacharje, H Joseph Yost, Luca Brunelli
Seamless (i.e., without unwanted DNA sequences) mutant bacterial artificial chromosomes (BACs) generated via recombination-mediated genetic engineering (recombineering) are better suited to study gene function compared to complementary DNA (cDNA) because they contain only the specific mutation and provide all the regulatory sequences required for in vivo gene expression. However, precisely mutated BACs are typically rare (∼1:1,000 to 1:100,000), making their isolation quite challenging. Although these BACs have been classically isolated by linking the mutation to additional genes, i...
April 3, 2017: Current Protocols in Molecular Biology
https://www.readbyqxmd.com/read/28369676/transcriptome-wide-identification-of-rna-binding-protein-binding-sites-using-photoactivatable-ribonucleoside-enhanced-crosslinking-immunoprecipitation-par-clip
#4
Henrike Maatz, Marcin Kolinski, Norbert Hubner, Markus Landthaler
RNA-binding proteins (RBPs) mediate important co- and post-transcriptional gene regulation by binding pre-mRNA in a sequence- and/or structure-specific manner. For a comprehensive understanding of RBP function, transcriptome-wide mapping of the RNA-binding sites is essential, and CLIP-seq methods have been developed to elucidate protein/RNA interactions at high resolution. CLIP-seq combines protein/RNA UV-crosslinking with immunoprecipitation (CLIP) followed by high-throughput sequencing of crosslinked RNA fragments...
April 3, 2017: Current Protocols in Molecular Biology
https://www.readbyqxmd.com/read/28369675/analysis-of-membrane-protein-interactions-with-a-bacterial-adenylate-cyclase-based-two-hybrid-bacth-technique
#5
Scot P Ouellette, Gouzel Karimova, Marilyne Davi, Daniel Ladant
The bacterial two-hybrid (BACTH, for "Bacterial Adenylate Cyclase-based Two-Hybrid") technique is a simple and fast genetic approach to analyze protein-protein interactions in vivo. In this system, the proteins of interest are genetically fused to two complementary fragments from the catalytic domain of Bordetella pertussis adenylate cyclase and co-expressed in strains of Escherichia coli deficient in adenylate cyclase. Association of the hybrid proteins restores synthesis of cyclic AMP (cAMP), which then triggers the expression of catabolic operons such as the lactose operon or the maltose regulon...
April 3, 2017: Current Protocols in Molecular Biology
https://www.readbyqxmd.com/read/28060411/scarless-cas9-assisted-recombineering-no-scar-in-escherichia-coli-an-easy-to-use-system-for-genome-editing
#6
Christopher R Reisch, Kristala L J Prather
The discovery and development of genome editing systems that leverage the site-specific DNA endonuclease system CRISPR/Cas9 has fundamentally changed the ease and speed of genome editing in many organisms. In eukaryotes, the CRISPR/Cas9 system utilizes a "guide" RNA to enable the Cas9 nuclease to make a double-strand break at a particular genome locus, which is repaired by non-homologous end joining (NHEJ) repair enzymes, often generating random mutations in the process. A specific alteration of the target genome can also be generated by supplying a DNA template in vivo with a desired mutation, which is incorporated by homology-directed repair...
January 5, 2017: Current Protocols in Molecular Biology
https://www.readbyqxmd.com/read/28060410/preparation-of-cell-cultures-and-vaccinia-virus-stocks
#7
Catherine A Cotter, Patricia L Earl, Linda S Wyatt, Bernard Moss
The culturing of cell lines used with vaccinia virus, both as monolayer and in suspension, is described. The preparation of chick embryo fibroblasts (CEF) is presented for use in the production of the highly attenuated and host range-restricted modified vaccinia virus Ankara (MVA) strain of vaccinia virus. Protocols for the preparation, titration, and trypsinization of vaccinia virus stocks, as well as viral DNA preparation and virus purification methods are also included. © 2017 by John Wiley & Sons, Inc.
January 5, 2017: Current Protocols in Molecular Biology
https://www.readbyqxmd.com/read/28060409/immunization-of-mice
#8
Tomás Maira-Litrán
The selection of an appropriate immunization strategy depends largely on the properties of an antigen, including its nature, purity, solubility, and availability. This unit describes the critical steps in the production of monoclonal and polyclonal antibodies against soluble molecules (such as proteins, peptides, polysaccharides, oligosaccharides, or hapten-conjugate vaccines), complex antigens (such as whole pathogens or outer membrane vesicles), and antigens embedded in or eluted from gel matrix following electrophoresis...
January 5, 2017: Current Protocols in Molecular Biology
https://www.readbyqxmd.com/read/28060408/next-generation-sequencing-for-identification-of-ems-induced-mutations-in-caenorhabditis-elegans
#9
Nicolas J Lehrbach, Fei Ji, Ruslan Sadreyev
Forward genetic analysis using chemical mutagenesis in model organisms is a powerful tool for investigation of molecular mechanisms in biological systems. In the nematode, Caenorhabditis elegans, mutagenesis screens using ethyl methanesulfonate (EMS) have led to important insights into genetic control of animal development and physiology. A major bottleneck to this approach is identification of the causative mutation underlying a phenotype of interest. In the past, this has required time-consuming genetic mapping experiments...
January 5, 2017: Current Protocols in Molecular Biology
https://www.readbyqxmd.com/read/28060407/mammalian-cell-tissue-culture-techniques
#10
Katy Phelan, Kristin M May
Cultured mammalian cells are used extensively in molecular biology studies. A number of special skills are required in order to preserve the structure, function, behavior, and biology of cells in culture. This appendix describes the basic skills required to maintain and preserve cell cultures: maintaining aseptic technique, preparing media with the appropriate characteristics, passaging, freezing and storage, recovering frozen stocks, and counting viable cells. © 2017 by John Wiley & Sons, Inc.
January 5, 2017: Current Protocols in Molecular Biology
https://www.readbyqxmd.com/read/28060406/a-simple-protoplast-based-method-for-screening-potent-artificial-mirna-for-maximal-gene-silencing-in-arabidopsis
#11
Nannan Zhang, Dandan Zhang, Jian-Feng Li
The unpredictability of in planta silencing efficiency of artificial micro RNAs (amiRNAs) has greatly limited the application of the amiRNA technology in basic and applied plant research. This unit describes a simple and robust method called the epitope-tagged protein-based amiRNA (ETPamir) screen for identifying the most effective amiRNA for silencing a particular Arabidopsis gene. After selecting three to four amiRNA candidates designed by the WMD3 web server for the target gene, the silencing efficiencies of the amiRNA candidates can be compared by co-expressing individual amiRNAs with the target mRNA encoding an epitope-tagged target protein in Arabidopsis mesophyll protoplasts...
January 5, 2017: Current Protocols in Molecular Biology
https://www.readbyqxmd.com/read/28060405/generation-of-recombinant-vaccinia-viruses
#12
Linda S Wyatt, Patricia L Earl, Bernard Moss
This unit describes how to infect cells with vaccinia virus and then transfect them with a plasmid-transfer vector or PCR fragment to generate a recombinant virus. Selection and screening methods used to isolate recombinant viruses and a method for the amplification of recombinant viruses are described. Finally, a method for live immunostaining that has been used primarily for detection of recombinant modified vaccinia virus Ankara (MVA) is presented. © 2017 by John Wiley & Sons, Inc.
January 5, 2017: Current Protocols in Molecular Biology
https://www.readbyqxmd.com/read/27723087/strand-specific-transcriptome-sequencing-using-smart-technology
#13
Magnolia Bostick, Nathalie Bolduc, Alisa Lehman, Andrew Farmer
Next-generation sequencing is empowering a deeper understanding of biology by enabling RNA expression analysis over the entire transcriptome with high sensitivity and a wide dynamic range. One powerful application within this field is stranded RNA sequencing (RNA-seq), which is necessary to distinguish overlapping genes and to conduct comprehensive annotation and quantification of long non-coding RNAs. Commonly used methods for generating strand-specific RNA-seq libraries are often complicated by protocols that require several rounds of enzymatic treatments and clean-up steps, making them time-intensive, insensitive, and unsuitable for processing several samples simultaneously...
October 10, 2016: Current Protocols in Molecular Biology
https://www.readbyqxmd.com/read/27723086/transcriptome-analysis-at-the-single-cell-level-using-smart-technology
#14
Rachel N Fish, Magnolia Bostick, Alisa Lehman, Andrew Farmer
RNA sequencing (RNA-seq) is a powerful method for analyzing cell state, with minimal bias, and has broad applications within the biological sciences. However, transcriptome analysis of seemingly homogenous cell populations may in fact overlook significant heterogeneity that can be uncovered at the single-cell level. The ultra-low amount of RNA contained in a single cell requires extraordinarily sensitive and reproducible transcriptome analysis methods. As next-generation sequencing (NGS) technologies mature, transcriptome profiling by RNA-seq is increasingly being used to decipher the molecular signature of individual cells...
October 10, 2016: Current Protocols in Molecular Biology
https://www.readbyqxmd.com/read/27723085/preparation-of-low-input-and-ligation-free-chip-seq-libraries-using-template-switching-technology
#15
Nathalie Bolduc, Alisa P Lehman, Andrew Farmer
Chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing (ChIP-seq) has become the gold standard for mapping of transcription factors and histone modifications throughout the genome. However, for ChIP experiments involving few cells or targeting low-abundance transcription factors, the small amount of DNA recovered makes ligation of adapters very challenging. In this unit, we describe a ChIP-seq workflow that can be applied to small cell numbers, including a robust single-tube and ligation-free method for preparation of sequencing libraries from sub-nanogram amounts of ChIP DNA...
October 10, 2016: Current Protocols in Molecular Biology
https://www.readbyqxmd.com/read/27723084/isolation-of-primary-fibroblast-culture-from-wildlife-the-panthera-onca-case-to-preserve-a-south-american-endangered-species
#16
Ana Cecilia Mestre-Citrinovitz, Adrián Jorge Sestelo, María Belén Ceballos, José Lino Barañao, Patricia Saragüeta
Cell line establishment of somatic cells is a valuable resource to preserve genetic material of rare, difficult-to-find, endangered and giant species like Jaguar (Panthera onca), the largest South American felid. This unit focuses on the isolation and culture of fibroblasts from Jaguar skin and muscle biopsies, and ear cartilage dissection immediately after death to preserve one of the several endangered species in this biome. These culture techniques enabled us to contribute 570 samples from 45 autochthonous and endangered species, including Jaguar...
October 10, 2016: Current Protocols in Molecular Biology
https://www.readbyqxmd.com/read/27723083/protein-interaction-profile-sequencing-pip-seq
#17
Shawn W Foley, Brian D Gregory
Every eukaryotic RNA transcript undergoes extensive post-transcriptional processing from the moment of transcription up through degradation. This regulation is performed by a distinct cohort of RNA-binding proteins which recognize their target transcript by both its primary sequence and secondary structure. Here, we describe protein interaction profile sequencing (PIP-seq), a technique that uses ribonuclease-based footprinting followed by high-throughput sequencing to globally assess both protein-bound RNA sequences and RNA secondary structure...
October 10, 2016: Current Protocols in Molecular Biology
https://www.readbyqxmd.com/read/27723082/gila-a-replacement-for-the-soft-agar-assay-that-permits-high-throughput-drug-and-genetic-screens-for-cellular-transformation
#18
Benjamin Izar, Asaf Rotem
For the last five decades, measuring the ability of cells to grow in soft agar has served as the gold standard assay for in vitro cellular transformation. Nevertheless, the soft agar colony formation assay is time consuming and ill-suited for high-throughput screens. This unit describes an equally qualitative and quantitative assay known as growth in low attachment or GILA. The GILA assay is suitable for high-throughput pharmacological or genetic screens and allows the simultaneous examination of multiple cell lines and experimental perturbations...
October 10, 2016: Current Protocols in Molecular Biology
https://www.readbyqxmd.com/read/27366888/detection-of-cell-proliferation-markers-by-immunofluorescence-staining-and-microscopy-imaging-in-paraffin-embedded-tissue-sections
#19
Seda Eminaga, Polakit Teekakirikul, Christine E Seidman, Jonathan G Seidman
This unit describes a step-by-step protocol to detect and quantify proliferating cells in paraffin-embedded tissue sections. Two well-established markers of proliferation (incorporation of BrdU into newly synthesized DNA and expression of the nuclear protein Ki67) are detected after antigen-retrieval and subsequent immunofluorescence staining and confocal microscopy. © 2016 by John Wiley & Sons, Inc.
July 1, 2016: Current Protocols in Molecular Biology
https://www.readbyqxmd.com/read/27038390/construction-of-a-sequencing-library-from-circulating-cell-free-dna
#20
Nan Fang, Dirk Löffert, Rumeysa Akinci-Tolun, Katja Heitz, Alexander Wolf
Circulating DNA is cell-free DNA (cfDNA) in serum or plasma that can be used for non-invasive prenatal testing, as well as cancer diagnosis, prognosis, and stratification. High-throughput sequence analysis of the cfDNA with next-generation sequencing technologies has proven to be a highly sensitive and specific method in detecting and characterizing mutations in cancer and other diseases, as well as aneuploidy during pregnancy. This unit describes detailed procedures to extract circulating cfDNA from human serum and plasma and generate sequencing libraries from a wide concentration range of circulating DNA...
April 1, 2016: Current Protocols in Molecular Biology
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