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Molecular Biology

Assaying the Effects of Splice Site Variants by Exon Trapping in a Mammalian Cell Line

Featured protocol,  Authors: Stuart W. Tompson
Stuart W. TompsonAffiliation: Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison, Madison, Wisconsin, USA
For correspondence: stompson@wisc.edu
Bio-protocol author page: a4509
 and Terri L. Young
Terri L. YoungAffiliation: Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison, Madison, Wisconsin, USA
Bio-protocol author page: a4510
date: 5/20/2017, 98 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2281.

Brief version appeared in J Clin Invest, Jul 2016
There are several in silico programs that endeavor to predict the functional impact of an individual’s sequence variation at splice donor/acceptor sites, but experimental confirmation is problematic without a source of RNA from the individual that carries the variant. With the aid of an exon trapping vector, such as pSPL3, an investigator can test whether a splice site sequence change leads to altered RNA splicing, through expression of reference and variant mini-genes in mammalian cells and analysis of the resultant RNA products.

Creating a RAW264.7 CRISPR-Cas9 Genome Wide Library

Featured protocol,  Authors: Brooke A Napier
Brooke A Napier Affiliation: Department of Microbiology and Immunology, Stanford School of Medicine, Stanford University, Stanford, CA, USA
Bio-protocol author page: a4566
 and Denise M Monack
Denise M MonackAffiliation: Department of Microbiology and Immunology, Stanford School of Medicine, Stanford University, Stanford, CA, USA
For correspondence: dmonack@stanford.edu
Bio-protocol author page: a4567
date: 5/20/2017, 112 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2320.

Brief version appeared in J Exp Med, Oct 2016
The bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 genome editing tools are used in mammalian cells to knock-out specific genes of interest to elucidate gene function. The CRISPR-Cas9 system requires that the mammalian cell expresses Cas9 endonuclease, guide RNA (gRNA) to lead the endonuclease to the gene of interest, and the PAM sequence that links the Cas9 to the gRNA. CRISPR-Cas9 genome wide libraries are used to screen the effect of each gene in the genome on the cellular phenotype of interest, in an unbiased high-throughput manner. In this protocol, we describe our method of creating a CRISPR-Cas9 genome wide library in a transformed murine macrophage cell-line (RAW264.7). We have employed this library to identify novel mediators in the caspase-11 cell death pathway (Napier et al., 2016); however, this library can then be used to screen the importance of specific genes in multiple murine macrophage cellular pathways.

Nucleosome Positioning Assay

Featured protocol,  Authors: Zhongliang Zhao
Zhongliang ZhaoAffiliation: Division of Molecular Biology of the Cell II, German Cancer Research Center, DKFZ-ZMBH Alliance, Im Neuenheimer Feld 581, 69120 Heidelberg, Germany
Bio-protocol author page: a4525
 and Holger Bierhoff
Holger BierhoffAffiliation 1: Department of Biochemistry, Institute of Biochemistry and Biophysics, Center for Molecular Biomedicine (CMB), Friedrich Schiller University Jena, Hans-Knöll-Str. 2, Jena, Germany
Affiliation 2: Leibniz-Institute on Aging – Fritz Lipmann Institute (FLI), Beutenbergstrasse 11, Jena, Germany
For correspondence: holger.bierhoff@uni-jena.de
Bio-protocol author page: a4526
date: 5/20/2017, 102 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2285.

Brief version appeared in Cell Rep, Mar 2016
The basic unit of chromatin is the nucleosome, a histone octamer with 147 base pairs of DNA wrapped around it. Positions of nucleosomes relative to each other and to DNA elements have a strong impact on chromatin structure and gene activity and are tightly regulated at multiple levels, i.e., DNA sequence, transcription factor binding, histone modifications and variants, and chromatin remodeling enzymes (Bell et al., 2011; Hughes and Rando, 2014). Nucleosome positions in cells or isolated nuclei can be detected by partial nuclease digestion of native or cross-linked chromatin followed by ligation-mediated polymerase chain reaction (LM-PCR) (McPherson et al., 1993; Soutoglou and Talianidis, 2002). This protocol describes a nucleosome positioning assay using Micrococcal Nuclease (MNase) digestion of formaldehyde-fixed chromatin followed by LM-PCR. We exemplify the nucleosome positioning assay for the promoter of genes encoding ribosomal RNA (rRNA genes or rDNA) in mice, which has two mutually exclusive configurations. The rDNA promoter harbors either an upstream nucleosome (NucU) covering nucleotides -157 to -2 relative to the transcription start site, or a downstream nucleosome (NucD) at position -132 to +22 (Li et al., 2006; Xie et al., 2012). Radioactive labeling of LM-PCR products followed by denaturing urea-polyacrylamide gel electrophoresis allows resolution and relative quantification of both configurations. As depicted in the diagram in Figure 1, the nucleosome positioning assay is a versatile low to medium throughput method to map discrete nucleosome positions with high precision in a semi-quantitative manner.

CRISPR-PCS Protocol for Chromosome Splitting and Splitting Event Detection in Saccharomyces cerevisiae

Featured protocol,  Authors: Yu Sasano
Yu SasanoAffiliation: Department of Applied Microbial Technology, Faculty of Biotechnology and Life Science, Sojo University, Kumamoto City, Japan
Bio-protocol author page: a4563
 and Satoshi Harashima
Satoshi HarashimaAffiliation: Department of Applied Microbial Technology, Faculty of Biotechnology and Life Science, Sojo University, Kumamoto City, Japan
For correspondence: harashima@bio.sojo-u.ac.jp
Bio-protocol author page: a4564
date: 5/20/2017, 93 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2306.

Brief version appeared in Sci Rep, Aug 2016
Chromosome engineering is an important technology with applications in basic biology and biotechnology. Chromosome splitting technology called PCS (PCR-mediated Chromosome Splitting) has already been developed as a fundamental chromosome engineering technology in the budding yeast. However, the splitting efficiency of PCS technology is not high enough to achieve multiple splitting at a time. This protocol describes a procedure for achieving simultaneous and multiple chromosome splits in the budding yeast Saccharomyces cerevisiae by a new technology called CRISPR-PCS. At least four independent sites in the genome can be split by one transformation. Total time and labor for obtaining a multiple split yeast strain is drastically reduced when compared with conventional PCS technology.

Ultradeep Pyrosequencing of Hepatitis C Virus to Define Evolutionary Phenotypes

Featured protocol,  Authors: Brendan A. Palmer
Brendan A. PalmerAffiliation: Molecular Virology Diagnostic & Research Laboratory, Department of Medicine, University College Cork, Cork, Ireland
Bio-protocol author page: a4519
Zoya Dimitrova
Zoya DimitrovaAffiliation: Division of Viral Hepatitis, Centers of Disease Control and Prevention, Atlanta, Georgia, USA
Bio-protocol author page: a4520
Pavel Skums
Pavel SkumsAffiliation: Division of Viral Hepatitis, Centers of Disease Control and Prevention, Atlanta, Georgia, USA
Bio-protocol author page: a4521
Orla Crosbie
Orla CrosbieAffiliation: Department of Gastroenterology, Cork University Hospital, Cork, Ireland
Bio-protocol author page: a4522
Elizabeth Kenny-Walsh
Elizabeth Kenny-WalshAffiliation: Department of Gastroenterology, Cork University Hospital, Cork, Ireland
Bio-protocol author page: a4523
 and Liam J. Fanning
Liam J. FanningAffiliation: Molecular Virology Diagnostic & Research Laboratory, Department of Medicine, University College Cork, Cork, Ireland
For correspondence: l.fanning@ucc.ie
Bio-protocol author page: a4524
date: 5/20/2017, 91 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2284.

Brief version appeared in J Virol, Dec 2015
Analysis of hypervariable regions (HVR) using pyrosequencing techniques is hampered by the ability of error correction algorithms to account for the heterogeneity of the variants present. Analysis of between-sample fluctuations to virome sub-populations, and detection of low frequency variants, are unreliable through the application of arbitrary frequency cut offs. Cumulatively this leads to an underestimation of genetic diversity. In the following technique we describe the analysis of Hepatitis C virus (HCV) HVR1 which includes the E1/E2 glycoprotein gene junction. This procedure describes the evolution of HCV in a treatment naïve environment, from 10 samples collected over 10 years, using ultradeep pyrosequencing (UDPS) performed on the Roche GS FLX titanium platform (Palmer et al., 2014). Initial clonal analysis of serum samples was used to inform downstream error correction algorithms that allowed for a greater sequence depth to be reached. PCR amplification of this region has been tested for HCV genotypes 1, 2, 3 and 4.

Endogenous C-terminal Tagging by CRISPR/Cas9 in Trypanosoma cruzi

Featured protocol,  Authors: Noelia Lander*
Noelia LanderAffiliation: Departamento de Patologia Clínica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Campinas, São Paulo, 13083, Brazil
For correspondence: noelia@uga.edu
Bio-protocol author page: a4559
Miguel A. Chiurillo*
Miguel A. ChiurilloAffiliation: Departamento de Patologia Clínica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Campinas, São Paulo, 13083, Brazil
Bio-protocol author page: a4560
Aníbal E. Vercesi
Aníbal E. VercesiAffiliation: Departamento de Patologia Clínica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Campinas, São Paulo, 13083, Brazil
Bio-protocol author page: a4561
 and Roberto Docampo
Roberto DocampoAffiliation 1: Departamento de Patologia Clínica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Campinas, São Paulo, 13083, Brazil
Affiliation 2: Center for Tropical and Emerging Global Diseases and Department of Cellular Biology, University of Georgia, Athens, Georgia, USA
Bio-protocol author page: a4562
 (*contributed equally to this work) date: 5/20/2017, 113 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2299.

Brief version appeared in J Biol Chem, Dec 2016
To achieve the C-terminal tagging of endogenous proteins in T. cruzi we use the Cas9/pTREX-n vector (Lander et al., 2015) to insert a specific tag sequence (3xHA or 3xc-Myc) at the 3’ end of a specific gene of interest (GOI). Chimeric sgRNA targeting the 3’ end of the GOI is PCR-amplified and cloned into Cas9/pTREX-n vector. Then a DNA donor molecule to induce DNA repair by homologous recombination is amplified. This donor sequence contains the tag sequence and a marker for antibiotic resistance, plus 100 bp homology arms corresponding to regions located right upstream of the stop codon and downstream of the Cas9 target site at the GOI locus. Vectors pMOTag23M (Oberholzer et al., 2006) or pMOHX1Tag4H (Lander et al., 2016b) are used as PCR templates for DNA donor amplification. Epimastigotes co-transfected with the sgRNA/Cas9/pTREX-n construct and the DNA donor cassette are then cultured for 5 weeks with antibiotics for selection of double resistant parasites. Endogenous gene tagging is finally verified by PCR and Western blot analysis.

A Method to Convert mRNA into a Guide RNA (gRNA) Library without Requiring Previous Bioinformatics Knowledge of the Organism

Featured protocol,  Author: Hiroshi Arakawa
Hiroshi ArakawaAffiliation: IFOM–FIRC Institute of Molecular Oncology Foundation, Milan, Italy
For correspondence: hiroshi.arakawa@ifom.eu
Bio-protocol author page: a4565
date: 5/20/2017, 93 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2319.

Brief version appeared in Sci Adv, Aug 2016
While the diversity of species represents a diversity of special biological abilities, many of the genes that encode those special abilities in a variety of species are untouched, leaving an untapped gold mine of genetic information; however, despite current advances in genome bioinformatics, annotation of that genetic information is incomplete in most species, except for well-established model organisms, such as human, mouse, or yeast. A guide RNA (gRNA) library using the clustered regularly interspersed palindromic repeats (CRISPR)/Cas9 (CRISPR-associated protein 9) system can be used for the phenotypic screening of uncharacterized genes by forward genetics. The construction of a gRNA library usually requires an abundance of chemically synthesized oligos designed from annotated genes; if one wants to convert mRNA into gRNA without prior knowledge of the target DNA sequences, the major challenges are finding the sequences flanking the protospacer adjacent motif (PAM) and cutting out the 20-bp fragment. Recently, I developed a molecular biology-based technique to convert mRNA into a gRNA library (Arakawa, 2016) (Figure 1). Here I describe the detailed protocol of how to construct a gRNA library from mRNA.

Biotinylated Micro-RNA Pull Down Assay for Identifying miRNA Targets

Featured protocol,  Authors: Pornima Phatak
Pornima PhatakAffiliation 1: Department of Surgery, University of Maryland School of Medicine, Baltimore, USA
Affiliation 2: Baltimore Veterans Affairs Medical Center, Baltimore, USA
Bio-protocol author page: a4420
 and James M Donahue
James M DonahueAffiliation 1: Department of Surgery, University of Maryland School of Medicine, Baltimore, USA
Affiliation 2: Baltimore Veterans Affairs Medical Center, Baltimore, USA
For correspondence: jdonahue@som.umaryland.edu
Bio-protocol author page: a4421
date: 5/5/2017, 202 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2253.

Brief version appeared in Oncogene, Apr 2016
microRNA (miRNA) directly associates with its target transcripts (mRNA). This protocol describes a method for detection of direct interaction between miRNA and mRNA. The result of interaction helps screening the specific target mRNAs for a miRNA.

Analysis of Replicative Intermediates of Adeno-associated Virus through Hirt Extraction and Southern Blotting

Featured protocol,  Authors: Martino Bardelli
Martino BardelliAffiliation: Department of Infectious Diseases, King's College London, London, United Kingdom
Present address: Jenner Institute, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom
Bio-protocol author page: a4283
Francisco Zarate-Perez
Francisco Zarate-PerezAffiliation: Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, Richmond VA, USA
Bio-protocol author page: a4284
Leticia Agundez
Leticia AgundezAffiliation: Department of Infectious Diseases, King's College London, London, United Kingdom
Present address: Department of Genetics, University College London Institute of Ophthalmology, London, United Kingdom
Bio-protocol author page: a4285
Nelly Jolinon
Nelly JolinonAffiliation: Department of Infectious Diseases, King’s College London, London, United Kingdom
Bio-protocol author page: a4486
R. Michael Linden
R. Michael LindenAffiliation: Department of Infectious Diseases, King's College London, London, United Kingdom
Present address: Genetic Medicine Institute, Pfizer Inc., London, United Kingdom
Bio-protocol author page: a4487
Carlos R. Escalante
Carlos R. EscalanteAffiliation: Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, Richmond, VA, USA
Bio-protocol author page: a4288
 and Els Henckaerts
Els HenckaertsAffiliation: Department of Infectious Diseases, King's College London, London, United Kingdom
For correspondence: els.henckaerts@kcl.ac.uk
Bio-protocol author page: a4289
date: 5/5/2017, 163 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2271.

Brief version appeared in J Virol, Aug 2016
Adeno-associated virus (AAV) is a small single-stranded DNA virus that requires the presence of a helper virus, such as adenovirus or herpes virus, to efficiently replicate its genome. AAV DNA is replicated by a rolling-hairpin mechanism (Ward, 2006), and during replication several DNA intermediates can be detected. This detailed protocol describes how to analyze the AAV DNA intermediates formed during AAV replication using a modified Hirt extract (Hirt, 1967) procedure and Southern blotting (Southern, 1975).

Conjugation Assay for Testing CRISPR-Cas Anti-plasmid Immunity in Staphylococci

Featured protocol,  Authors: Forrest C. Walker
Forrest C. WalkerAffiliation: Department of Biological Sciences, University of Alabama, Tuscaloosa, USA
Bio-protocol author page: a4490
 and Asma Hatoum-Aslan
Asma Hatoum-AslanAffiliation: Department of Biological Sciences, University of Alabama, Tuscaloosa, USA
For correspondence: ahatoum@ua.edu
Bio-protocol author page: a4491
date: 5/5/2017, 218 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2293.

Brief version appeared in J Bacteriol, Jan 2014
CRISPR-Cas is a prokaryotic adaptive immune system that prevents uptake of mobile genetic elements such as bacteriophages and plasmids. Plasmid transfer between bacteria is of particular clinical concern due to increasing amounts of antibiotic resistant pathogens found in humans as a result of transfer of resistance plasmids within and between species. Testing the ability of CRISPR-Cas systems to block plasmid transfer in various conditions or with CRISPR-Cas mutants provides key insights into the functionality and mechanisms of CRISPR-Cas as well as how antibiotic resistance spreads within bacterial communities. Here, we describe a method for quantifying the impact of CRISPR-Cas on the efficiency of plasmid transfer by conjugation. While this method is presented in Staphylococcus species, it could be more broadly used for any conjugative prokaryote.

Evaluation of Plasmid Stability by Negative Selection in Gram-negative Bacteria

Featured protocol,  Authors: Damián Lobato Márquez
Damián Lobato MárquezAffiliation: Department of Medicine, Imperial College London, London, UK
For correspondence: d.marquez@imperial.ac.uk
Bio-protocol author page: a4462
 and Laura Molina García
Laura Molina GarcíaAffiliation: Department of Cell and Developmental Biology, University College London, London, UK
Bio-protocol author page: a1812
date: 5/5/2017, 139 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2261.

Brief version appeared in Front Mol Biosci, Oct 2016
Plasmid stability can be measured using antibiotic-resistance plasmid derivatives by positive selection. However, highly stable plasmids are below the sensitivity range of these assays. To solve this problem we describe a novel, highly sensitive method to measure plasmid stability based on the selection of plasmid-free cells following elimination of plasmid-containing cells. The assay proposed here is based on an aph-parE cassette. When synthesized in the cell, the ParE toxin induces cell death. ParE synthesis is controlled by a rhamnose-inducible promoter. When bacteria carrying the aph-parE module are grown in media containing rhamnose as the only carbon source, ParE is synthesized and plasmid-containing cells are eliminated. Kanamycin resistance (aph) is further used to confirm the absence of the plasmid in rhamnose grown bacteria.

Assaying the Effects of Splice Site Variants by Exon Trapping in a Mammalian Cell Line

Authors: Stuart W. Tompson
Stuart W. TompsonAffiliation: Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison, Madison, Wisconsin, USA
For correspondence: stompson@wisc.edu
Bio-protocol author page: a4509
 and Terri L. Young
Terri L. YoungAffiliation: Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison, Madison, Wisconsin, USA
Bio-protocol author page: a4510
date: 5/20/2017, 98 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2281.

[Abstract] There are several in silico programs that endeavor to predict the functional impact of an individual’s sequence variation at splice donor/acceptor sites, but experimental confirmation is problematic without a source of RNA from the individual that carries the variant. With the aid of an exon trapping vector, such as pSPL3, an investigator can test ...

Creating a RAW264.7 CRISPR-Cas9 Genome Wide Library

Authors: Brooke A Napier
Brooke A Napier Affiliation: Department of Microbiology and Immunology, Stanford School of Medicine, Stanford University, Stanford, CA, USA
Bio-protocol author page: a4566
 and Denise M Monack
Denise M MonackAffiliation: Department of Microbiology and Immunology, Stanford School of Medicine, Stanford University, Stanford, CA, USA
For correspondence: dmonack@stanford.edu
Bio-protocol author page: a4567
date: 5/20/2017, 112 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2320.

[Abstract] The bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 genome editing tools are used in mammalian cells to knock-out specific genes of interest to elucidate gene function. The CRISPR-Cas9 system requires that the mammalian cell expresses Cas9 endonuclease, guide RNA (gRNA) to lead the endonuclease to the gene of interest, ...

Nucleosome Positioning Assay

Authors: Zhongliang Zhao
Zhongliang ZhaoAffiliation: Division of Molecular Biology of the Cell II, German Cancer Research Center, DKFZ-ZMBH Alliance, Im Neuenheimer Feld 581, 69120 Heidelberg, Germany
Bio-protocol author page: a4525
 and Holger Bierhoff
Holger BierhoffAffiliation 1: Department of Biochemistry, Institute of Biochemistry and Biophysics, Center for Molecular Biomedicine (CMB), Friedrich Schiller University Jena, Hans-Knöll-Str. 2, Jena, Germany
Affiliation 2: Leibniz-Institute on Aging – Fritz Lipmann Institute (FLI), Beutenbergstrasse 11, Jena, Germany
For correspondence: holger.bierhoff@uni-jena.de
Bio-protocol author page: a4526
date: 5/20/2017, 102 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2285.

[Abstract] The basic unit of chromatin is the nucleosome, a histone octamer with 147 base pairs of DNA wrapped around it. Positions of nucleosomes relative to each other and to DNA elements have a strong impact on chromatin structure and gene activity and are tightly regulated at multiple levels, i.e., DNA sequence, transcription factor binding, histone modifications ...

CRISPR-PCS Protocol for Chromosome Splitting and Splitting Event Detection in Saccharomyces cerevisiae

Authors: Yu Sasano
Yu SasanoAffiliation: Department of Applied Microbial Technology, Faculty of Biotechnology and Life Science, Sojo University, Kumamoto City, Japan
Bio-protocol author page: a4563
 and Satoshi Harashima
Satoshi HarashimaAffiliation: Department of Applied Microbial Technology, Faculty of Biotechnology and Life Science, Sojo University, Kumamoto City, Japan
For correspondence: harashima@bio.sojo-u.ac.jp
Bio-protocol author page: a4564
date: 5/20/2017, 93 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2306.

[Abstract] Chromosome engineering is an important technology with applications in basic biology and biotechnology. Chromosome splitting technology called PCS (PCR-mediated Chromosome Splitting) has already been developed as a fundamental chromosome engineering technology in the budding yeast. However, the splitting efficiency of PCS technology is not high enough ...

Ultradeep Pyrosequencing of Hepatitis C Virus to Define Evolutionary Phenotypes

Authors: Brendan A. Palmer
Brendan A. PalmerAffiliation: Molecular Virology Diagnostic & Research Laboratory, Department of Medicine, University College Cork, Cork, Ireland
Bio-protocol author page: a4519
Zoya Dimitrova
Zoya DimitrovaAffiliation: Division of Viral Hepatitis, Centers of Disease Control and Prevention, Atlanta, Georgia, USA
Bio-protocol author page: a4520
Pavel Skums
Pavel SkumsAffiliation: Division of Viral Hepatitis, Centers of Disease Control and Prevention, Atlanta, Georgia, USA
Bio-protocol author page: a4521
Orla Crosbie
Orla CrosbieAffiliation: Department of Gastroenterology, Cork University Hospital, Cork, Ireland
Bio-protocol author page: a4522
Elizabeth Kenny-Walsh
Elizabeth Kenny-WalshAffiliation: Department of Gastroenterology, Cork University Hospital, Cork, Ireland
Bio-protocol author page: a4523
 and Liam J. Fanning
Liam J. FanningAffiliation: Molecular Virology Diagnostic & Research Laboratory, Department of Medicine, University College Cork, Cork, Ireland
For correspondence: l.fanning@ucc.ie
Bio-protocol author page: a4524
date: 5/20/2017, 91 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2284.

[Abstract] Analysis of hypervariable regions (HVR) using pyrosequencing techniques is hampered by the ability of error correction algorithms to account for the heterogeneity of the variants present. Analysis of between-sample fluctuations to virome sub-populations, and detection of low frequency variants, are unreliable through the application of arbitrary frequency ...

Endogenous C-terminal Tagging by CRISPR/Cas9 in Trypanosoma cruzi

Authors: Noelia Lander*
Noelia LanderAffiliation: Departamento de Patologia Clínica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Campinas, São Paulo, 13083, Brazil
For correspondence: noelia@uga.edu
Bio-protocol author page: a4559
Miguel A. Chiurillo*
Miguel A. ChiurilloAffiliation: Departamento de Patologia Clínica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Campinas, São Paulo, 13083, Brazil
Bio-protocol author page: a4560
Aníbal E. Vercesi
Aníbal E. VercesiAffiliation: Departamento de Patologia Clínica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Campinas, São Paulo, 13083, Brazil
Bio-protocol author page: a4561
 and Roberto Docampo
Roberto DocampoAffiliation 1: Departamento de Patologia Clínica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Campinas, São Paulo, 13083, Brazil
Affiliation 2: Center for Tropical and Emerging Global Diseases and Department of Cellular Biology, University of Georgia, Athens, Georgia, USA
Bio-protocol author page: a4562
 (*contributed equally to this work) date: 5/20/2017, 113 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2299.

[Abstract] To achieve the C-terminal tagging of endogenous proteins in T. cruzi we use the Cas9/pTREX-n vector (Lander et al., 2015) to insert a specific tag sequence (3xHA or 3xc-Myc) at the 3’ end of a specific gene of interest (GOI). Chimeric sgRNA targeting the 3’ end of the GOI is PCR-amplified and cloned into Cas9/pTREX-n vector. Then a DNA donor molecule ...

A Method to Convert mRNA into a Guide RNA (gRNA) Library without Requiring Previous Bioinformatics Knowledge of the Organism

Author: Hiroshi Arakawa
Hiroshi ArakawaAffiliation: IFOM–FIRC Institute of Molecular Oncology Foundation, Milan, Italy
For correspondence: hiroshi.arakawa@ifom.eu
Bio-protocol author page: a4565
date: 5/20/2017, 93 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2319.

[Abstract] While the diversity of species represents a diversity of special biological abilities, many of the genes that encode those special abilities in a variety of species are untouched, leaving an untapped gold mine of genetic information; however, despite current advances in genome bioinformatics, annotation of that genetic information is incomplete in ...

Biotinylated Micro-RNA Pull Down Assay for Identifying miRNA Targets

Authors: Pornima Phatak
Pornima PhatakAffiliation 1: Department of Surgery, University of Maryland School of Medicine, Baltimore, USA
Affiliation 2: Baltimore Veterans Affairs Medical Center, Baltimore, USA
Bio-protocol author page: a4420
 and James M Donahue
James M DonahueAffiliation 1: Department of Surgery, University of Maryland School of Medicine, Baltimore, USA
Affiliation 2: Baltimore Veterans Affairs Medical Center, Baltimore, USA
For correspondence: jdonahue@som.umaryland.edu
Bio-protocol author page: a4421
date: 5/5/2017, 202 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2253.

[Abstract] microRNA (miRNA) directly associates with its target transcripts (mRNA). This protocol describes a method for detection of direct interaction between miRNA and mRNA. The result of interaction helps screening the specific target mRNAs for a miRNA. ...

Analysis of Replicative Intermediates of Adeno-associated Virus through Hirt Extraction and Southern Blotting

Authors: Martino Bardelli
Martino BardelliAffiliation: Department of Infectious Diseases, King's College London, London, United Kingdom
Present address: Jenner Institute, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom
Bio-protocol author page: a4283
Francisco Zarate-Perez
Francisco Zarate-PerezAffiliation: Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, Richmond VA, USA
Bio-protocol author page: a4284
Leticia Agundez
Leticia AgundezAffiliation: Department of Infectious Diseases, King's College London, London, United Kingdom
Present address: Department of Genetics, University College London Institute of Ophthalmology, London, United Kingdom
Bio-protocol author page: a4285
Nelly Jolinon
Nelly JolinonAffiliation: Department of Infectious Diseases, King’s College London, London, United Kingdom
Bio-protocol author page: a4486
R. Michael Linden
R. Michael LindenAffiliation: Department of Infectious Diseases, King's College London, London, United Kingdom
Present address: Genetic Medicine Institute, Pfizer Inc., London, United Kingdom
Bio-protocol author page: a4487
Carlos R. Escalante
Carlos R. EscalanteAffiliation: Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, Richmond, VA, USA
Bio-protocol author page: a4288
 and Els Henckaerts
Els HenckaertsAffiliation: Department of Infectious Diseases, King's College London, London, United Kingdom
For correspondence: els.henckaerts@kcl.ac.uk
Bio-protocol author page: a4289
date: 5/5/2017, 163 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2271.

[Abstract] Adeno-associated virus (AAV) is a small single-stranded DNA virus that requires the presence of a helper virus, such as adenovirus or herpes virus, to efficiently replicate its genome. AAV DNA is replicated by a rolling-hairpin mechanism (Ward, 2006), and during replication several DNA intermediates can be detected. This detailed protocol describes ...

Conjugation Assay for Testing CRISPR-Cas Anti-plasmid Immunity in Staphylococci

Authors: Forrest C. Walker
Forrest C. WalkerAffiliation: Department of Biological Sciences, University of Alabama, Tuscaloosa, USA
Bio-protocol author page: a4490
 and Asma Hatoum-Aslan
Asma Hatoum-AslanAffiliation: Department of Biological Sciences, University of Alabama, Tuscaloosa, USA
For correspondence: ahatoum@ua.edu
Bio-protocol author page: a4491
date: 5/5/2017, 218 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2293.

[Abstract] CRISPR-Cas is a prokaryotic adaptive immune system that prevents uptake of mobile genetic elements such as bacteriophages and plasmids. Plasmid transfer between bacteria is of particular clinical concern due to increasing amounts of antibiotic resistant pathogens found in humans as a result of transfer of resistance plasmids within and between species. ...
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[Bio101] Plasmid DNA Extraction from E. coli Using Alkaline Lysis Method

Author: Fanglian He
Fanglian HeAffiliation: Department of Biology, University of Pennsylvania, Philadelphia, USA
For correspondence: fanglian09@gmail.com
Bio-protocol author page: a9
date: 2/5/2011, 90358 views, 31 Q&A
DOI: https://doi.org/10.21769/BioProtoc.30.

[Abstract] This is a quick and efficient way to extract E. coli plasmid DNA without using commercial kits....

[Bio101] E. coli Genomic DNA Extraction Updates
The author made some updates (highlighted in blue) to the protocol on 09/12/2016.

Author: Fanglian He
Fanglian HeAffiliation: Department of Biology, University of Pennsylvania, Philadelphia, USA
Bio-protocol author page: a9
date: 7/20/2011, 83488 views, 46 Q&A
DOI: https://doi.org/10.21769/BioProtoc.97.

[Abstract] This protocol uses phenol/chloroform method to purify genomic DNA without using commercial kits....

[Bio101] DNA Molecular Weight Calculation

Author: Fanglian He date: 3/20/2011, 36280 views, 7 Q&A
DOI: https://doi.org/10.21769/BioProtoc.46.

[Abstract] This method is to roughly estimate DNA molecular weight. One of its applications is to calculate the ratio of vector to insert in a ligation reaction (please see Standard DNA Cloning protocol).
Anhydrous molecular weight of each nucleotide is (see reference 1):
A= 313.21
T= 304.2
C= 289.18
G=329.21
For rough ...





[Bio101] Infiltration of Nicotiana benthamiana Protocol for Transient Expression via Agrobacterium

Author: Xiyan Li
Xiyan LiAffiliation: Department of Genetics, Stanford University, Stanford, USA
For correspondence: lixiyan@stanford.edu
Bio-protocol author page: a13
date: 7/20/2011, 31987 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.95.

[Abstract] Transient expression in tobacco plant (Nicotiana benthamiana) is used to determine the subcellular location of a protein of interest when tagged with a reporter such as green fluorescent protein (GFP), or to mass produce proteins without making transgenic plants. The root tumor bacteria, Agrobacteria, ...

[Bio101] Calcium Phosphate Transfection of Eukaryotic Cells

Author: Yanling Chen
Yanling ChenAffiliation: Department of Immunology, The Scripps Research Institute, La Jolla, USA
For correspondence: ylchen@scripps.edu
Bio-protocol author page: a27
date: 2/5/2012, 28112 views, 2 Q&A
DOI: https://doi.org/10.21769/BioProtoc.86.

[Abstract] Transfection of DNA into cells is an indispensible protocol in molecular biology. While plenty of lipid-based transfection reagents are commercially available nowadays, a quick, simple, efficient and inexpensive method is to transfect eukaryotic cells via calcium phosphate co-precipitation with DNA ...

C2C12 Myoblasts

Author: Lili Jing
Lili JingAffiliation: Department of Cell and Molecular Biology, University of Pennsylvania, Philadelphia, USA
For correspondence: lilijingcn@gmail.com
Bio-protocol author page: a38
date: 5/20/2012, 27726 views, 11 Q&A
DOI: https://doi.org/10.21769/BioProtoc.172.

[Abstract] C2C12 myoblasts are commonly used in biomedical laboratories as an in vitro system to study muscle development and differentiation. This protocol explains the basic procedures of culture, transfection and differentiation of C2C12 myoblast cells....

[Bio101] A General EMSA (Gel-shift) Protocol

Author: Ran Chen
Ran ChenAffiliation: Department of Genetics, Stanford University, Stanford, USA
For correspondence: rcchen@jfkbio.com
Bio-protocol author page: a34
date: 2/5/2011, 25207 views, 3 Q&A
DOI: https://doi.org/10.21769/BioProtoc.24.

[Abstract] An electrophoretic mobility shift assay (EMSA), also referred to as mobility shift electrophoresis, a gel shift assay, gel mobility shift assay, band shift assay, or gel retardation assay, is a common technique used to study protein-DNA or protein-RNA interactions. The control lane (the DNA/RNA probe ...

[Bio101] Standard DNA Cloning

Author: Fanglian He date: 4/5/2011, 24889 views, 4 Q&A
DOI: https://doi.org/10.21769/BioProtoc.52.

[Abstract] This protocol describes general cloning steps from preparation of both vector and insert DNA to the ligation reaction....

[Bio101] Lentivirus Production

Author: Nabila Aboulaich date: 3/5/2011, 23596 views, 6 Q&A
DOI: https://doi.org/10.21769/BioProtoc.39.

[Abstract] Lentivirus is a common tool used to introduce a gene into mammalian or other animal cells.This protocol is to produce lentivirus stocks from hairpin-pLKO.1 plasmid....

Analysis of Protein Stability by the Cycloheximide Chase Assay

Authors: Shih-Han Kao
Shih-Han KaoAffiliation: Research Center for Tumor Medical Science, China Medical University, Taichung, Taiwan
Bio-protocol author page: a1920
Wen-Lung Wang
Wen-Lung WangAffiliation: Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan
Bio-protocol author page: a1921
Chi-Yuan Chen
Chi-Yuan ChenAffiliation: Department of Nutrition and Health Sciences, Chang Gung University of Science and Technology, Taoyuan, Taiwan
Bio-protocol author page: a1922
Yih-Leong Chang
Yih-Leong ChangAffiliation: Department of Pathology and Graduate Institute of Pathology, National Taiwan University, Taipei, Taiwan
Bio-protocol author page: a1923
Yi-Ying Wu
Yi-Ying WuAffiliation: Graduate Institute of Clinical Medicine, National Cheng Kung University, Taipei, Taiwan
Bio-protocol author page: a1924
Yi-Ting Wang
Yi-Ting WangAffiliation 1: Chemical Biology and Molecular Biophysics Program, Academia Sinica, Taipei, Taiwan
Affiliation 2: Institute of Chemistry, Academia Sinica, Taipei, Taiwan
Affiliation 3: Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan
Bio-protocol author page: a1925
Shu-Ping Wang
Shu-Ping WangAffiliation: Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, New York, USA
Bio-protocol author page: a1926
Alexey I Nesvizhskii
Alexey I NesvizhskiiAffiliation 1: Department of Pathology, University of Michigan, Ann Arbor, USA
Affiliation 2: Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, USA
Bio-protocol author page: a1927
Yu-Ju Chen
Yu-Ju ChenAffiliation 1: Chemical Biology and Molecular Biophysics Program, Academia Sinica, Taipei, Taiwan
Affiliation 2: Institute of Chemistry, Academia Sinica, Taipei, Taiwan
Bio-protocol author page: a1928
Tse-Ming Hong
Tse-Ming HongAffiliation: Graduate Institute of Clinical Medicine, National Cheng Kung University, Taipei, Taiwan
Bio-protocol author page: a1929
 and Pan-Chyr Yang
Pan-Chyr YangAffiliation 1: Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan
Affiliation 2: Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan
Affiliation 3: NTU Center of Genomic Medicine, National Taiwan University, Taipei, Taiwan
Present address: Department of Internal Medicine, National Taiwan University College of Medicine, Taipei, Taiwan
For correspondence: pcyang@ntu.edu.tw
Bio-protocol author page: a574
date: 1/5/2015, 22635 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1374.

[Abstract] Comparison of protein stability in eukaryotic cells has been achieved by cycloheximide, which is an inhibitor of protein biosynthesis due to its prevention in translational elongation. It is broadly used in cell biology in terms of determining the half-life of a given protein and has gained much popularity ...
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