Viruses

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    Protocols in Current Issue
    Detachment Procedure of Bacteria from Atmospheric Particles for Flow-cytometry Counting
    Authors:  Carolina M. Araya, Alberto Cazorla and Isabel Reche, date: 06/20/2019, view: 577, Q&A: 0
    [Abstract] The protocol separates bacteria from atmospheric particles, obtaining with greater precision their abundance in the atmospheric deposition. This procedure is similar to the one used to separate bacteria in streambed sediments. The detachment procedure consists of a chemical treatment with sodium pyrophosphate and Tween 20 and a physical treatment ...
    A Flow Cytometric Method to Determine Transfection Efficiency
    [Abstract] Mammalian cell transfection is a powerful technique commonly used in molecular biology to express exogenous DNA or RNA in cells and study gene and protein function. Although several transfection strategies have been developed, there is a wide variation with regards to transfection efficiency, cell toxicity and reproducibility. Thus, a sensitive ...
    Next-generation Sequencing of the DNA Virome from Fecal Samples
    Authors:  Cynthia L. Monaco and Douglas S. Kwon, date: 03/05/2017, view: 5291, Q&A: 0
    [Abstract] Herein we describe a detailed protocol for DNA virome analysis of low input human stool samples (Monaco et al., 2016). This protocol is divided into four main steps: 1) stool samples are pulverized to evenly distribute microbial matter; 2) stool is enriched for virus-like particles and DNA is extracted by phenol-chloroform; 3) purified ...
    TCRβ Clonotype Analysis of EBV and CMV-specific Human CD8+ T Cells
    Authors:  Nening M. Nanlohy, Dan Koning, Esther D. Quakkelaar and Debbie van Baarle, date: 10/05/2015, view: 4516, Q&A: 0
    [Abstract] This protocol describes the quantification of all expressed T-cell antigen receptor (TCR) gene products within sorted (by flow cytometry) EBV and CMV-specific memory CD8+ T-cell populations using a template-switch anchored reverse transcription polymerase chain reaction (RT-PCR).
    Determining the Relative Fitness Score of Mutant Viruses in a Population Using Illumina Paired-end Sequencing and Regression Analysis
    Authors:  Hangfei Qi, C. Anders Olson, Nicholas C. Wu, Yushen Du and Ren Sun, date: 05/20/2015, view: 5597, Q&A: 0
    [Abstract] Recent advances in DNA sequencing capacity to accurately quantify the copy number of individual variants in a large and diverse population allows in parallel determination of the phenotypic effects caused by each genetic modification. This systematic profiling approach is a combination of forward and reverse genetics, which we refer to as ...
    Construction of FWPV Chimaeric MVA
    Authors:  Karen R. Buttigieg and Michael A. Skinner, date: 01/05/2015, view: 5305, Q&A: 1
    [Abstract] Construction of chimaeric MVA is a useful tool with which to study gene function of related viruses. The protocol given here describes MVA chimaeras containing genes from Fowlpox virus (FWPV), although this can be applied to DNA derived from other organisms. There are a number of steps required to make the chimaeric MVA: 1) Purification of viral ...
    Fluorometric Estimation of Viral Thermal Stability
    Authors:  Vamseedhar Rayaprolu, Shannon Kruse, Ravi Kant, Navid Movahed, Dewey Brooke and Brian Bothner, date: 08/05/2014, view: 6807, Q&A: 1
    [Abstract] Differential Scanning Fluorimetry (DSF) is a rapid, economical, and a straightforward technique for estimating the thermal stability of proteins. The principle involves the binding of a fluorescent dye to thermally exposed hydrophobic pockets of a protein. The dyes used in this technique are highly fluorescent in a non-polar environment and are ...
    Fluorescence Microscopy Analysis of Drug Effect on Autophagosome Formation
    Authors:  Metodi Stankov, Diana Panayotova-Dimitrova, Martin Leverkus and Georg Behrens, date: 04/05/2014, view: 7470, Q&A: 0
    [Abstract] The autophagy protein, LC3 represents a reliable characteristic marker for autophagosomal structures. The initial LC3 is processed by the cysteine protease autophagy-related gene 4 (Atg4) at its C terminus in order to create LC3-I generally localized in the cytoplasm. Afterwards LC3-I is conjugated with phosphatidylethanolamine (PE) to become ...
    Helicase Assays
    Authors:  Xin Wang, Jing Li, Jason Diaz and Jianxin You, date: 03/20/2014, view: 9521, Q&A: 0
    [Abstract] Helicases are a class of enzymes which are motor proteins using energy derived from ATP hydrolysis to move directionally along a nucliec acid phosphodiester backbone (such as DNA, RNA and DNA-RNA hybrids) and separate two annealed nucleic acid strands. Many cellular processes, such as transcription, DNA replication, recombination and DNA repair ...
    Biochemical Assays for MTase Activity
    Authors:  Yu Chen and Deyin Guo, date: 01/20/2014, view: 4933, Q&A: 0
    [Abstract] Methyltransferase (MTase) transfers a methyl group (-CH3) from the donor S-adenosyl-L-methionine (AdoMet or SAM) to biologically active molecules such as hormones, neurotransmitters, lipids, proteins and nucleic acids. The addition of a methyl group causes a change in the physicochemical properties of the molecules. The mRNA cap structure is ...



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