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RNA-protein UV-crosslinking Assay

Featured protocol,  Authors: Dipak Kumar Poria
Dipak Kumar PoriaAffiliation: Department of Biological Sciences, Indian Institute of Science Education and Research, Kolkata, India
Bio-protocol author page: a4215
 and Partho Sarothi Ray
Partho Sarothi RayAffiliation: Department of Biological Sciences, Indian Institute of Science Education and Research, Kolkata, India
For correspondence: psray@iiserkol.ac.in
Bio-protocol author page: a4216
date: 3/20/2017, 176 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2193.

Brief version appeared in Oncogene, Mar 2016
RNA-protein interactions play a crucial role in every aspect of RNA metabolism, and also plays a major role in post-transcriptional gene regulation. RNA-binding proteins have been implicated in viral gene expression (Ray and Das, 2002) and microRNA-mediated gene regulation (Poria et al., 2016). Here we have described the protocol which (1) covalently links transiently interacting RNA-protein complexes by UV crosslinking, (2) removes the unprotected RNA by RNase digestion and (3) detects the RNA-protein complexes by SDS-PAGE analysis. This protocol provides a rapid and reliable means to directly assay RNA-protein interactions and their kinetics using purified proteins and also help in identifying novel RNA-protein interactions

In silico Analysis and Site-directed Mutagenesis of Promoters

Featured protocol,  Authors: Salah Boudjadi
Salah BoudjadiAffiliation: Cancer Molecular Pathology Section, Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, USA
For correspondence: Salah.Boudjadi@nih.gov
Bio-protocol author page: a4261
 and Jean-Francois Beaulieu
Jean-Francois BeaulieuAffiliation: Laboratory of Intestinal Physiopathology, Department of Anatomy and Cell Biology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Canada
For correspondence: Jean-Francois.Beaulieu@USherbrooke.ca
Bio-protocol author page: a4262
date: 3/20/2017, 135 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2181.

Brief version appeared in Oncogene, Mar 2016
In normal as in cancerous cells, gene expression is tightly regulated by transcription factors, which are responsible for up- or down-regulation of thousands of targets involved in different cell processes. Transcription factors can directly regulate the expression of genes by binding to specific DNA sequences known as response elements. Identification of these response elements is important to characterize targets of transcription factors in order to understand their contribution to gene regulation. Here, we describe In silico analysis coupled to selected mutagenesis and promoter gene reporter assay procedures to identify and analyze response elements in the proximal promoter sequence of genes.

Measurement of RNA-induced PKR Activation in vitro

Featured protocol,  Author: Kobe C. Yuen
Kobe C. YuenAffiliation: Stowers Institute for Medical Research, Kansas City, MO, USA
Present address: 1 DNA Way, South San Francisco, USA
For correspondence: yuenc4@gene.com
Bio-protocol author page: a4260
date: 3/20/2017, 141 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2178.

Brief version appeared in Cell Rep, Jan 2016
Protein kinase R (PKR) is one of the key RNA-activated sensors for innate immunity. PKR is activated by pathogenic or aberrant RNAs such as short double-stranded RNAs or those with imperfect secondary structures, as well as a reduction in the amount and number of RNA modifications. Activation of PKR may be an underlying mechanism for the pathogenesis of human diseases. In this protocol, I describe a method for studying levels of RNA-induced PKR activation in vitro.

Determination of Adeno-associated Virus Rep DNA Binding Using Fluorescence Anisotropy

Featured protocol,  Authors: 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
Vishaka Santosh
Vishaka SantoshAffiliation: Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, Richmond VA, USA
Bio-protocol author page: a4286
Martino Bardelli
Martino BardelliAffiliation: Department of Infectious Diseases, King’s College London, London, United Kingdom
Bio-protocol author page: a4283
Leticia Agundez
Leticia AgundezAffiliation: Department of Infectious Diseases, King’s College London, London, United Kingdom
Bio-protocol author page: a4285
R. Michael Linden
R. Michael LindenAffiliation: Department of Infectious Diseases, King’s College London, London, United Kingdom
Bio-protocol author page: a4287
Els Henckaerts
Els HenckaertsAffiliation: Department of Infectious Diseases, King’s College London, London, United Kingdom
Bio-protocol author page: a4289
 and Carlos R. Escalante
Carlos R. EscalanteAffiliation: Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, Richmond VA, USA
For correspondence: carlos.escalante@vcuhealth.org
Bio-protocol author page: a4288
date: 3/20/2017, 93 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2194.

Brief version appeared in J Virol, Jul 2016
Quantitative measurement of proteins binding to DNA is a requisite to fully characterize the structural determinants of complex formation necessary to understand the DNA transactions that regulate cellular processes. Here we describe a detailed protocol to measure binding affinity of the adeno-associated virus (AAV) Rep68 protein for the integration site AAVS1 using fluorescent anisotropy. This protocol can be used to measure the binding constants of any DNA binding protein provided the substrate DNA is fluorescently labeled.

Ribosomal RNA N-glycosylase Activity Assay of Ribosome-inactivating Proteins

Featured protocol,  Authors: Rosario Iglesias
Rosario IglesiasAffiliation: Department of Biochemistry and Molecular Biology and Physiology, Faculty of Sciences, University of Valladolid, Valladolid, Spain
Bio-protocol author page: a4233
Lucía Citores
Lucía CitoresAffiliation: Department of Biochemistry and Molecular Biology and Physiology, Faculty of Sciences, University of Valladolid, Valladolid, Spain
Bio-protocol author page: a4234
 and José M. Ferreras
José M. FerrerasAffiliation: Department of Biochemistry and Molecular Biology and Physiology, Faculty of Sciences, University of Valladolid, Valladolid, Spain
For correspondence: rosario@bio.uva.es
Bio-protocol author page: a4232
date: 3/20/2017, 106 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2180.

Brief version appeared in Mol Plant Pathol, Feb 2016
Ribosome-inactivating proteins (RIPs) are enzymes that irreversibly inactivate ribosomes as a consequence of their N-glycosylase (EC 3.2.2.22) activity. The enzyme cleaves the N-glycosidic bond between the adenine No. 4324 from the 28S rRNA and its ribose in rat ribosomes (or the equivalent adenine in sensitive ribosomes from other organisms). This adenine is located in the α-sarcin-ricin loop (SRL) that is crucial for anchoring the elongation factor (EF) G and EF2 on the ribosome during mRNA-tRNA translocation in prokaryotes and eukaryotes, respectively. RIPs have been isolated mainly from plants and examples of these proteins are ricin or Pokeweed Antiviral Protein (PAP). These proteins, either alone or as a part of immunotoxins, are useful tools for cancer therapy. The following protocol describes a method to detect the RNA fragment released when the RIP-treated apurinic RNA from rabbit reticulocyte lysate is incubated in the presence of acid aniline by electrophoresis on polyacrylamide gels. The fragment released (Endo’s fragment) is diagnostic of the action of RIPs.

Next-generation Sequencing of the DNA Virome from Fecal Samples

Featured protocol,  Authors: Cynthia L. Monaco
Cynthia L. MonacoAffiliation: Department of Medicine, Washington University School of Medicine, St. Louis, USA
For correspondence: cmonaco@wustl.edu
Bio-protocol author page: a4038
 and Douglas S. Kwon
Douglas S. KwonAffiliation 1: Ragon Institute of MGH, MIT, and Harvard, Cambridge, USA
Affiliation 2: Division of Infectious Diseases, Massachusetts General Hospital and Harvard Medical School, Boston, USA
Bio-protocol author page: a4039
date: 3/5/2017, 199 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2159.

Brief version appeared in Cell Host Microbe, Mar 2016
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 DNA is multiple-strand displacement amplified (MDA) and fragmented; and 4) libraries are constructed and sequenced using Illumina Miseq. Subsequent sequence analysis for viral sequence identification should be sensitive but stringent.

Ca2+ Measurements in Mammalian Cells with Aequorin-based Probes

Featured protocol,  Authors: Anna Tosatto
Anna TosattoAffiliation: Department of Biomedical Sciences, University of Padua, Padua, Italy
Bio-protocol author page: a4128
Rosario Rizzuto
Rosario RizzutoAffiliation: Department of Biomedical Sciences, University of Padua, Padua, Italy
For correspondence: rosario.rizzuto@unipd.it
Bio-protocol author page: a4129
 and Cristina Mammucari
Cristina MammucariAffiliation: Department of Biomedical Sciences, University of Padua, Padua, Italy
For correspondence: cristina.mammucari@unipd.it
Bio-protocol author page: a4130
date: 3/5/2017, 213 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2155.

Brief version appeared in EMBO Mol Med, May 2016
Aequorin is a Ca2+ sensitive photoprotein suitable to measure intracellular Ca2+ transients in mammalian cells. Thanks to recombinant cDNAs expression, aequorin can be specifically targeted to various subcellular compartments, thus allowing an accurate measurement of Ca2+ uptake and release of different intracellular organelles. Here, we describe how to use this probe to measure cytosolic Ca2+ levels and mitochondrial Ca2+ uptake in mammalian cells.

Knock-in Blunt Ligation Utilizing CRISPR/Cas9

Featured protocol,  Authors: Jonathan M. Geisinger
Jonathan M. GeisingerAffiliation: Department of Biology, Stanford University, Stanford, USA
For correspondence: jonmg54@stanford.edu
Bio-protocol author page: a4146
 and Michele P. Calos
Michele P. CalosAffiliation: Department of Genetics, Stanford University, Stanford, USA
Bio-protocol author page: a4147
date: 3/5/2017, 197 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2163.

Brief version appeared in Nucleic Acids Res, May 2016
The incorporation of the CRISPR/Cas9 bacterial immune system into the genetic engineering toolbox has led to the development of several new methods for genome manipulation (Auer et al., 2014; Byrne et al., 2015). We took advantage of the ability of Cas9 to generate blunt-ended double-strand breaks (Jinek et al., 2012) to introduce exogenous DNA in a highly precise manner through the exploitation of non-homologous end-joining DNA repair machinery (Geisinger et al., 2016). This protocol has been successfully applied to traditional immortalized cell lines and human induced pluripotent stem cells. Here we present a generalized protocol for knock-in blunt ligation, using HEK293 cells as an example.

Polyethylene Glycol-mediated Transformation of Drechmeria coniospora

Featured protocol,  Authors: Le D. He
Le D. HeAffiliation: Aix Marseille Univ, CNRS, INSERM, CIML, Centre d’Immunologie de Marseille-Luminy, Marseille, France
Bio-protocol author page: a4195
 and Jonathan J. Ewbank
Jonathan J. EwbankAffiliation: Aix Marseille Univ, CNRS, INSERM, CIML, Centre d’Immunologie de Marseille-Luminy, Marseille, France
For correspondence: ewbank@ciml.univ-mrs.fr
Bio-protocol author page: a4196
date: 3/5/2017, 193 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2157.

Brief version appeared in PLoS Genet, May 2016
Drechmeria coniospora is a nematophagous fungus and potential biocontrol agent. It belongs to the Ascomycota. It is related to Hirsutella minnesotensis, another nematophagous fungus but, phylogenetically, it is currently closest to the truffle parasite Tolypocladium ophioglossoides. Together with its natural host, Caenorhabditis elegans, it is used to study host-pathogen interactions. Here, we report a polyethylene glycol-mediated transformation method (Turgeon et al., 2010; Ochman et al., 1988) for this fungus. The protocol can be used to generate both knock-in or knock-out strains (Lebrigand et al., 2016).

Multiplexed GuideRNA-expression to Efficiently Mutagenize Multiple Loci in Arabidopsis by CRISPR-Cas9

Featured protocol,  Authors: Julia Schumacher
Julia SchumacherAffiliation 1: Institute for Biochemistry and Biology, University of Potsdam, Potsdam, Germany
Affiliation 2: Department for Plant Cell and Molecular Biology, Institute for Biology, Humboldt-Universität zu Berlin, Berlin, Germany
Bio-protocol author page: a4201
Kerstin Kaufmann
Kerstin KaufmannAffiliation: Department for Plant Cell and Molecular Biology, Institute for Biology, Humboldt-Universität zu Berlin, Berlin, Germany
Bio-protocol author page: a4202
 and Wenhao Yan
Wenhao YanAffiliation 1: Institute for Biochemistry and Biology, University of Potsdam, Potsdam, Germany
Affiliation 2: Department for Plant Cell and Molecular Biology, Institute for Biology, Humboldt-Universität zu Berlin, Berlin, Germany
For correspondence: yanwen@uni-potsdam.de
Bio-protocol author page: a4203
date: 3/5/2017, 230 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2166.

Brief version appeared in Plant Methods, Apr 2016
Since the discovery of the CRISPR (clustered regularly interspaced short palindromic repeats)-associated protein (Cas) as an efficient tool for genome editing in plants (Li et al., 2013; Shan et al., 2013; Nekrasov et al., 2013), a large variety of applications, such as gene knock-out, knock-in or transcriptional regulation, has been published. So far, the generation of multiple mutants in plants involved tedious crossing or mutagenesis followed by time-consuming screening of huge populations and the use of the Cas9-system appeared a promising method to overcome these issues. We designed a binary vector that combines both the coding sequence of the codon optimized Streptococcus pyogenes Cas9 nuclease under the control of the Arabidopsis thaliana UBIQUITIN10 (UBQ10)-promoter and guideRNA (gRNA) expression cassettes driven by the A. thaliana U6-promoter for efficient multiplex editing in Arabidopsis (Yan et al., 2016). Here, we describe a step-by-step protocol to cost-efficiently generate the binary vector containing multiple gRNAs and the Cas9 nuclease based on classic cloning procedure.

RNA-protein UV-crosslinking Assay

Authors: Dipak Kumar Poria
Dipak Kumar PoriaAffiliation: Department of Biological Sciences, Indian Institute of Science Education and Research, Kolkata, India
Bio-protocol author page: a4215
 and Partho Sarothi Ray
Partho Sarothi RayAffiliation: Department of Biological Sciences, Indian Institute of Science Education and Research, Kolkata, India
For correspondence: psray@iiserkol.ac.in
Bio-protocol author page: a4216
date: 3/20/2017, 176 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2193.

[Abstract] RNA-protein interactions play a crucial role in every aspect of RNA metabolism, and also plays a major role in post-transcriptional gene regulation. RNA-binding proteins have been implicated in viral gene expression (Ray and Das, 2002) and microRNA-mediated gene regulation (Poria et al., 2016). Here we have described the protocol which (1) covalently ...

In silico Analysis and Site-directed Mutagenesis of Promoters

Authors: Salah Boudjadi
Salah BoudjadiAffiliation: Cancer Molecular Pathology Section, Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, USA
For correspondence: Salah.Boudjadi@nih.gov
Bio-protocol author page: a4261
 and Jean-Francois Beaulieu
Jean-Francois BeaulieuAffiliation: Laboratory of Intestinal Physiopathology, Department of Anatomy and Cell Biology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Canada
For correspondence: Jean-Francois.Beaulieu@USherbrooke.ca
Bio-protocol author page: a4262
date: 3/20/2017, 135 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2181.

[Abstract] In normal as in cancerous cells, gene expression is tightly regulated by transcription factors, which are responsible for up- or down-regulation of thousands of targets involved in different cell processes. Transcription factors can directly regulate the expression of genes by binding to specific DNA sequences known as response elements. Identification ...

Measurement of RNA-induced PKR Activation in vitro

Author: Kobe C. Yuen
Kobe C. YuenAffiliation: Stowers Institute for Medical Research, Kansas City, MO, USA
Present address: 1 DNA Way, South San Francisco, USA
For correspondence: yuenc4@gene.com
Bio-protocol author page: a4260
date: 3/20/2017, 141 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2178.

[Abstract] Protein kinase R (PKR) is one of the key RNA-activated sensors for innate immunity. PKR is activated by pathogenic or aberrant RNAs such as short double-stranded RNAs or those with imperfect secondary structures, as well as a reduction in the amount and number of RNA modifications. Activation of PKR may be an underlying mechanism for the pathogenesis ...

Determination of Adeno-associated Virus Rep DNA Binding Using Fluorescence Anisotropy

Authors: 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
Vishaka Santosh
Vishaka SantoshAffiliation: Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, Richmond VA, USA
Bio-protocol author page: a4286
Martino Bardelli
Martino BardelliAffiliation: Department of Infectious Diseases, King’s College London, London, United Kingdom
Bio-protocol author page: a4283
Leticia Agundez
Leticia AgundezAffiliation: Department of Infectious Diseases, King’s College London, London, United Kingdom
Bio-protocol author page: a4285
R. Michael Linden
R. Michael LindenAffiliation: Department of Infectious Diseases, King’s College London, London, United Kingdom
Bio-protocol author page: a4287
Els Henckaerts
Els HenckaertsAffiliation: Department of Infectious Diseases, King’s College London, London, United Kingdom
Bio-protocol author page: a4289
 and Carlos R. Escalante
Carlos R. EscalanteAffiliation: Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, Richmond VA, USA
For correspondence: carlos.escalante@vcuhealth.org
Bio-protocol author page: a4288
date: 3/20/2017, 93 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2194.

[Abstract] Quantitative measurement of proteins binding to DNA is a requisite to fully characterize the structural determinants of complex formation necessary to understand the DNA transactions that regulate cellular processes. Here we describe a detailed protocol to measure binding affinity of the adeno-associated virus (AAV) Rep68 protein for the integration ...

Ribosomal RNA N-glycosylase Activity Assay of Ribosome-inactivating Proteins

Authors: Rosario Iglesias
Rosario IglesiasAffiliation: Department of Biochemistry and Molecular Biology and Physiology, Faculty of Sciences, University of Valladolid, Valladolid, Spain
Bio-protocol author page: a4233
Lucía Citores
Lucía CitoresAffiliation: Department of Biochemistry and Molecular Biology and Physiology, Faculty of Sciences, University of Valladolid, Valladolid, Spain
Bio-protocol author page: a4234
 and José M. Ferreras
José M. FerrerasAffiliation: Department of Biochemistry and Molecular Biology and Physiology, Faculty of Sciences, University of Valladolid, Valladolid, Spain
For correspondence: rosario@bio.uva.es
Bio-protocol author page: a4232
date: 3/20/2017, 106 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2180.

[Abstract] Ribosome-inactivating proteins (RIPs) are enzymes that irreversibly inactivate ribosomes as a consequence of their N-glycosylase (EC 3.2.2.22) activity. The enzyme cleaves the N-glycosidic bond between the adenine No. 4324 from the 28S rRNA and its ribose in rat ribosomes (or the equivalent adenine in sensitive ribosomes from other organisms). This ...

Next-generation Sequencing of the DNA Virome from Fecal Samples

Authors: Cynthia L. Monaco
Cynthia L. MonacoAffiliation: Department of Medicine, Washington University School of Medicine, St. Louis, USA
For correspondence: cmonaco@wustl.edu
Bio-protocol author page: a4038
 and Douglas S. Kwon
Douglas S. KwonAffiliation 1: Ragon Institute of MGH, MIT, and Harvard, Cambridge, USA
Affiliation 2: Division of Infectious Diseases, Massachusetts General Hospital and Harvard Medical School, Boston, USA
Bio-protocol author page: a4039
date: 3/5/2017, 199 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2159.

[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 DNA is multiple-strand ...

Ca2+ Measurements in Mammalian Cells with Aequorin-based Probes

Authors: Anna Tosatto
Anna TosattoAffiliation: Department of Biomedical Sciences, University of Padua, Padua, Italy
Bio-protocol author page: a4128
Rosario Rizzuto
Rosario RizzutoAffiliation: Department of Biomedical Sciences, University of Padua, Padua, Italy
For correspondence: rosario.rizzuto@unipd.it
Bio-protocol author page: a4129
 and Cristina Mammucari
Cristina MammucariAffiliation: Department of Biomedical Sciences, University of Padua, Padua, Italy
For correspondence: cristina.mammucari@unipd.it
Bio-protocol author page: a4130
date: 3/5/2017, 213 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2155.

[Abstract] Aequorin is a Ca2+ sensitive photoprotein suitable to measure intracellular Ca2+ transients in mammalian cells. Thanks to recombinant cDNAs expression, aequorin can be specifically targeted to various subcellular compartments, thus allowing an accurate measurement of Ca2+ uptake and release of different intracellular organelles. Here, we describe how ...

Knock-in Blunt Ligation Utilizing CRISPR/Cas9

Authors: Jonathan M. Geisinger
Jonathan M. GeisingerAffiliation: Department of Biology, Stanford University, Stanford, USA
For correspondence: jonmg54@stanford.edu
Bio-protocol author page: a4146
 and Michele P. Calos
Michele P. CalosAffiliation: Department of Genetics, Stanford University, Stanford, USA
Bio-protocol author page: a4147
date: 3/5/2017, 197 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2163.

[Abstract] The incorporation of the CRISPR/Cas9 bacterial immune system into the genetic engineering toolbox has led to the development of several new methods for genome manipulation (Auer et al., 2014; Byrne et al., 2015). We took advantage of the ability of Cas9 to generate blunt-ended double-strand breaks (Jinek et al., 2012) to introduce exogenous DNA in ...

Polyethylene Glycol-mediated Transformation of Drechmeria coniospora

Authors: Le D. He
Le D. HeAffiliation: Aix Marseille Univ, CNRS, INSERM, CIML, Centre d’Immunologie de Marseille-Luminy, Marseille, France
Bio-protocol author page: a4195
 and Jonathan J. Ewbank
Jonathan J. EwbankAffiliation: Aix Marseille Univ, CNRS, INSERM, CIML, Centre d’Immunologie de Marseille-Luminy, Marseille, France
For correspondence: ewbank@ciml.univ-mrs.fr
Bio-protocol author page: a4196
date: 3/5/2017, 193 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2157.

[Abstract] Drechmeria coniospora is a nematophagous fungus and potential biocontrol agent. It belongs to the Ascomycota. It is related to Hirsutella minnesotensis, another nematophagous fungus but, phylogenetically, it is currently closest to the truffle parasite Tolypocladium ophioglossoides. Together with its natural host, Caenorhabditis elegans, it is used ...

Multiplexed GuideRNA-expression to Efficiently Mutagenize Multiple Loci in Arabidopsis by CRISPR-Cas9

Authors: Julia Schumacher
Julia SchumacherAffiliation 1: Institute for Biochemistry and Biology, University of Potsdam, Potsdam, Germany
Affiliation 2: Department for Plant Cell and Molecular Biology, Institute for Biology, Humboldt-Universität zu Berlin, Berlin, Germany
Bio-protocol author page: a4201
Kerstin Kaufmann
Kerstin KaufmannAffiliation: Department for Plant Cell and Molecular Biology, Institute for Biology, Humboldt-Universität zu Berlin, Berlin, Germany
Bio-protocol author page: a4202
 and Wenhao Yan
Wenhao YanAffiliation 1: Institute for Biochemistry and Biology, University of Potsdam, Potsdam, Germany
Affiliation 2: Department for Plant Cell and Molecular Biology, Institute for Biology, Humboldt-Universität zu Berlin, Berlin, Germany
For correspondence: yanwen@uni-potsdam.de
Bio-protocol author page: a4203
date: 3/5/2017, 230 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2166.

[Abstract] Since the discovery of the CRISPR (clustered regularly interspaced short palindromic repeats)-associated protein (Cas) as an efficient tool for genome editing in plants (Li et al., 2013; Shan et al., 2013; Nekrasov et al., 2013), a large variety of applications, such as gene knock-out, knock-in or transcriptional regulation, has been published. So ...
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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, 85875 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, 78982 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, 34760 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, 30896 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, 27173 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, 26867 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, 24031 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, 23956 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, 22841 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....

In vitro Protein Kinase Assay

Author: Yuehua Wei
Yuehua WeiAffiliation: Department of Pharmacology, Cancer Institute of New Jersey, UMDNJ Robert Wood Johnson Medical School, Piscataway, USA
For correspondence: weiyh.sjtu.edu@gmail.com
Bio-protocol author page: a49
date: 6/5/2012, 21457 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.212.

[Abstract] This protocol will describe experimental procedures for an in vitro kinase assay of the yeast protein kinase Sch9. This protocol can be tailored to detect kinase activity of other yeast protein kinase....
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