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Dot Blot Analysis of N6-methyladenosine RNA Modification Levels

Featured protocol,  Authors: Lisha Shen
Lisha ShenAffiliation: Temasek Life Sciences Laboratory and Department of Biological Sciences, National University of Singapore, Singapore
Bio-protocol author page: a4000
Zhe Liang
Zhe Liang Affiliation: Temasek Life Sciences Laboratory and Department of Biological Sciences, National University of Singapore, Singapore
Bio-protocol author page: a4001
 and Hao Yu
Hao YuAffiliation: Temasek Life Sciences Laboratory and Department of Biological Sciences, National University of Singapore, Singapore
For correspondence: dbsyuhao@nus.edu.sg
Bio-protocol author page: a4002
date: 1/5/2017, 85 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2095.

Brief version appeared in Dev Cell, Jul 2016
N6-methyladenosine (m6A) is the most prevalent internal modification of eukaryotic messenger RNA (mRNA). The total amount of m6A can be detected by several methods, such as dot blot analysis using specific m6A antibodies and quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) (Fu et al., 2014; Shen et al., 2016). Here we describe the method for fast detection of total m6A levels in mRNA by dot blot analysis using a specific m6A antibody.

Measuring Procaspase-8 and -10 Processing upon Apoptosis Induction

Featured protocol,  Authors: Sabine Pietkiewicz
Sabine PietkiewiczAffiliation: Translational Inflammation Research, Institute of Experimental Internal Medicine, Otto von Guericke University Magdeburg, Medical Faculty, Magdeburg, Germany
Present address: Medical advisory service of social health insurance, Essen, Germany
Bio-protocol author page: a3934
Clara Wolfe
Clara WolfeAffiliation: Translational Inflammation Research, Institute of Experimental Internal Medicine, Otto von Guericke University Magdeburg, Medical Faculty, Magdeburg, Germany
Bio-protocol author page: a3935
Jörn H. Buchbinder
Jörn H. BuchbinderAffiliation: Translational Inflammation Research, Institute of Experimental Internal Medicine, Otto von Guericke University Magdeburg, Medical Faculty, Magdeburg, Germany
Bio-protocol author page: a3936
 and Inna N. Lavrik
Inna N. LavrikAffiliation 1: Translational Inflammation Research, Institute of Experimental Internal Medicine, Otto von Guericke University Magdeburg, Medical Faculty, Magdeburg, Germany
Affiliation 2: Federal research center Institute of Cytology and Genetics, Novosibirsk, Russia
For correspondence: inna.lavrik@med.ovgu.de
Bio-protocol author page: a3937
date: 1/5/2017, 97 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2081.

Brief version appeared in Cell Death Differ, Apr 2016
Apoptosis or programmed cell death is important for multicellular organisms to keep cell homeostasis and for the clearance of mutated or infected cells. Apoptosis can be induced by intrinsic or extrinsic stimuli. The first event in extrinsic apoptosis is the formation of the Death-Inducing Signalling Complex (DISC), where the initiator caspases-8 and -10 are fully activated by several proteolytic cleavage steps and induce the caspase cascade leading to apoptotic cell death. Analysing the processing of procaspases-8 and -10 by Western blot is a commonly used method to study the induction of apoptosis by death receptor stimulation. To analyse procaspase-8 and -10 cleavage, cells are stimulated with a death ligand for different time intervals, lysed and subjected to Western blot analysis using anti-caspase-8 and anti-caspase-10 antibodies. This allows monitoring the caspase cleavage products and thereby induction of apoptosis.

Fluorescence in situ Localization of Gene Expression Using a lacZ Reporter in the Heterocyst-forming Cyanobacterium Anabaena variabilis

Featured protocol,  Authors: Brenda S. Pratte
Brenda S. PratteAffiliation: Department of Biology, University of Missouri – St. Louis, St. Louis, MO, USA
Bio-protocol author page: a3944
 and Teresa Thiel
Teresa ThielAffiliation: Department of Biology, University of Missouri – St. Louis, St. Louis, MO, USA
For correspondence: thiel@umsl.edu
Bio-protocol author page: a3945
date: 1/5/2017, 71 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2084.

Brief version appeared in Mol Microbiol, Jun 2016
One of the most successful fluorescent proteins, used as a reporter of gene expression in many bacterial, plant and animals, is green fluorescent protein and its modified forms, which also function well in cyanobacteria. However, these fluorescent proteins do not allow rapid and economical quantitation of the reporter gene product, as does the popular reporter gene lacZ, encoding the enzyme β-galactosidase. We provide here a protocol for the in situ localization of β-galactosidase activity in cyanobacterial cells. This allows the same strain to be used for both a simple, quantitative, colorimetric assay with the substrate ortho-nitrophenyl-β-galactoside (ONPG) and for sensitive, fluorescence-based, cell-type localization of gene expression using 5-dodecanolyaminofluorescein di-β-D-galactopyranoside (C12-FDG).

Efficient AAV-mediated Gene Targeting Using 2A-based Promoter-trap System

Featured protocol,  Authors: Sivasundaram Karnan
Sivasundaram KarnanAffiliation: Department of Biochemistry, Aichi Medical University School of Medicine, Nagakute, Aichi, Japan
Bio-protocol author page: a3860
Akinobu Ota
Akinobu OtaAffiliation: Department of Biochemistry, Aichi Medical University School of Medicine, Nagakute, Aichi, Japan
Bio-protocol author page: a3861
Yuko Konishi
Yuko KonishiAffiliation: Department of Biochemistry, Aichi Medical University School of Medicine, Nagakute, Aichi, Japan
Bio-protocol author page: a3862
Md Wahiduzzaman
Md WahiduzzamanAffiliation: Department of Biochemistry, Aichi Medical University School of Medicine, Nagakute, Aichi, Japan
Bio-protocol author page: a3863
Shinobu Tsuzuki
Shinobu TsuzukiAffiliation: Department of Biochemistry, Aichi Medical University School of Medicine, Nagakute, Aichi, Japan
Bio-protocol author page: a3864
Yoshitaka Hosokawa
Yoshitaka HosokawaAffiliation: Department of Biochemistry, Aichi Medical University School of Medicine, Nagakute, Aichi, Japan
Bio-protocol author page: a3865
 and Hiroyuki Konishi
Hiroyuki KonishiAffiliation: Department of Biochemistry, Aichi Medical University School of Medicine, Nagakute, Aichi, Japan
For correspondence: hkonishi@aichi-med-u.ac.jp
Bio-protocol author page: a3866
date: 12/20/2016, 160 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2058.

Brief version appeared in Nucleic Acids Res, Apr 2016
Adeno-associated virus (AAV)-based targeting vectors have 1-4-log higher gene targeting efficiencies compared with plasmid-based targeting vectors. The efficiency of AAV-mediated gene targeting is further increased by introducing a promoter-trap system into targeting vectors. In addition, we found that the use of ribosome-skipping 2A peptide rather than commonly used internal ribosome entry site (IRES) in the promoter-trap system results in significantly higher AAV-mediated gene targeting efficiencies (Karnan et al., 2016). In this protocol, we describe the procedures for AAV-mediated gene targeting exploiting 2A for promoter trapping, including the construction of a targeting vector based on the platform plasmid pAAV-2Aneo or pAAV-2Aneo v2, production of AAV particles, infection of cells with resulting AAV-based targeting vectors, and isolation and verification of gene-targeted cell clones.

Affinity Pulldown of Biotinylated RNA for Detection of Protein-RNA Complexes

Featured protocol,  Authors: Amaresh C Panda
Amaresh C PandaAffiliation: Laboratory of Genetics and Genomics, National Institute on Aging, National Institutes of Health, 251 Bayview Blvd., Baltimore, United States
For correspondence: amaresh.panda@nih.gov
Bio-protocol author page: a3875
Jennifer L. Martindale
Jennifer L. Martindale Affiliation: Laboratory of Genetics and Genomics, National Institute on Aging, National Institutes of Health, 251 Bayview Blvd., Baltimore, United States
Bio-protocol author page: a3880
 and Myriam Gorospe
Myriam GorospeAffiliation: Laboratory of Genetics and Genomics, National Institute on Aging, National Institutes of Health, 251 Bayview Blvd., Baltimore, United States
Bio-protocol author page: a3881
date: 12/20/2016, 228 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2062.

Brief version appeared in Nucleic Acids Res, Mar 2016
RNA-binding proteins (RBPs) have recently emerged as crucial players in the regulation of gene expression. The interactions of RBPs with target mRNAs control the levels of gene products by altering different regulatory steps, including pre-mRNA splicing and maturation, nuclear mRNA export, and mRNA stability and translation (Glisovic et al., 2008). There are several methodologies available today to identify RNAs bound to specific RBPs; some detect only recombinant molecules in vitro, others detect recombinant and endogenous molecules, while others detect only endogenous molecules. Examples include systematic evolution of ligands by exponential enrichment (SELEX), biotinylated RNA pulldown assay, RNA immunoprecipitation (RIP) assay, electrophoretic mobility shift assay (EMSA), RNA footprinting analysis, and various UV crosslinking and immunoprecipitation (CLIP) methods such as CLIP, PAR-CLIP, and iCLIP (Popova et al., 2015). Here, we describe a simple and informative method to study and identify the RNA region of interaction between an RBP and its target transcript (Panda et al., 2014 and 2016). Its reproducibility and ease of use make this protocol a fast and useful method to identify interactions between RBPs and specific RNAs.

Gene Expression Analysis of Sorted Cells by RNA-seq in Drosophila Intestine

Featured protocol,  Authors: Jun Chen
Jun ChenAffiliation: National Institute of Biological Sciences, Beijing, China
Bio-protocol author page: a3929
Jia Li
Jia LiAffiliation: National Institute of Biological Sciences, Beijing, China
Bio-protocol author page: a16
Huaiwei Huang
Huaiwei HuangAffiliation: National Institute of Biological Sciences, Beijing, China
Bio-protocol author page: a3931
 and Rongwen Xi
Rongwen XiAffiliation: National Institute of Biological Sciences, Beijing, China
For correspondence: xirongwen@nibs.ac.cn
Bio-protocol author page: a3932
date: 12/20/2016, 185 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2079.

Brief version appeared in eLife, Jun 2016
RNA sequencing (RNA-seq) has become a popular method for profiling gene expression. Among many applications, one common purpose is to identify differentially expressed genes and pathways in different biological or pathological conditions. This protocol provides detailed procedure for RNA-seq analysis of ~250,000 sorted Drosophila intestinal cells (Chen et al., 2016), in which RNA amplification is not required.

Single-step Marker Switching in Schizosaccharomyces pombe Using a Lithium Acetate Transformation Protocol

Featured protocol,  Authors: Simon David Brown
Simon David BrownAffiliation: Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK
Bio-protocol author page: a3916
 and Alexander Lorenz
Alexander LorenzAffiliation: Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK
For correspondence: a.lorenz@abdn.ac.uk
Bio-protocol author page: a3917
date: 12/20/2016, 316 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2075.

Brief version appeared in Yeast, Dec 2015
The ability to utilize different selectable markers for tagging or mutating multiple genes in Schizosaccharomyces pombe is hampered by the historical use of only two selectable markers, ura4+ and kanMX6; the latter conferring resistance to the antibiotic G418 (geneticin). More markers have been described recently, but introducing these into yeast cells often requires strain construction from scratch. To overcome this problem we and other groups have created transformation cassettes with flanking homologies to ura4+ and kanMX6 which enable an efficient and time-saving way to exchange markers in existing mutated or tagged fission yeast strains.

Dot Blot Analysis of N6-methyladenosine RNA Modification Levels

Authors: Lisha Shen
Lisha ShenAffiliation: Temasek Life Sciences Laboratory and Department of Biological Sciences, National University of Singapore, Singapore
Bio-protocol author page: a4000
Zhe Liang
Zhe Liang Affiliation: Temasek Life Sciences Laboratory and Department of Biological Sciences, National University of Singapore, Singapore
Bio-protocol author page: a4001
 and Hao Yu
Hao YuAffiliation: Temasek Life Sciences Laboratory and Department of Biological Sciences, National University of Singapore, Singapore
For correspondence: dbsyuhao@nus.edu.sg
Bio-protocol author page: a4002
date: 1/5/2017, 85 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2095.

[Abstract] N6-methyladenosine (m6A) is the most prevalent internal modification of eukaryotic messenger RNA (mRNA). The total amount of m6A can be detected by several methods, such as dot blot analysis using specific m6A antibodies and quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) (Fu et al., 2014; Shen et al., 2016). Here we describe ...

Measuring Procaspase-8 and -10 Processing upon Apoptosis Induction

Authors: Sabine Pietkiewicz
Sabine PietkiewiczAffiliation: Translational Inflammation Research, Institute of Experimental Internal Medicine, Otto von Guericke University Magdeburg, Medical Faculty, Magdeburg, Germany
Present address: Medical advisory service of social health insurance, Essen, Germany
Bio-protocol author page: a3934
Clara Wolfe
Clara WolfeAffiliation: Translational Inflammation Research, Institute of Experimental Internal Medicine, Otto von Guericke University Magdeburg, Medical Faculty, Magdeburg, Germany
Bio-protocol author page: a3935
Jörn H. Buchbinder
Jörn H. BuchbinderAffiliation: Translational Inflammation Research, Institute of Experimental Internal Medicine, Otto von Guericke University Magdeburg, Medical Faculty, Magdeburg, Germany
Bio-protocol author page: a3936
 and Inna N. Lavrik
Inna N. LavrikAffiliation 1: Translational Inflammation Research, Institute of Experimental Internal Medicine, Otto von Guericke University Magdeburg, Medical Faculty, Magdeburg, Germany
Affiliation 2: Federal research center Institute of Cytology and Genetics, Novosibirsk, Russia
For correspondence: inna.lavrik@med.ovgu.de
Bio-protocol author page: a3937
date: 1/5/2017, 97 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2081.

[Abstract] Apoptosis or programmed cell death is important for multicellular organisms to keep cell homeostasis and for the clearance of mutated or infected cells. Apoptosis can be induced by intrinsic or extrinsic stimuli. The first event in extrinsic apoptosis is the formation of the Death-Inducing Signalling Complex (DISC), where the initiator caspases-8 and ...

Fluorescence in situ Localization of Gene Expression Using a lacZ Reporter in the Heterocyst-forming Cyanobacterium Anabaena variabilis

Authors: Brenda S. Pratte
Brenda S. PratteAffiliation: Department of Biology, University of Missouri – St. Louis, St. Louis, MO, USA
Bio-protocol author page: a3944
 and Teresa Thiel
Teresa ThielAffiliation: Department of Biology, University of Missouri – St. Louis, St. Louis, MO, USA
For correspondence: thiel@umsl.edu
Bio-protocol author page: a3945
date: 1/5/2017, 71 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2084.

[Abstract] One of the most successful fluorescent proteins, used as a reporter of gene expression in many bacterial, plant and animals, is green fluorescent protein and its modified forms, which also function well in cyanobacteria. However, these fluorescent proteins do not allow rapid and economical quantitation of the reporter gene product, as does the popular ...

Efficient AAV-mediated Gene Targeting Using 2A-based Promoter-trap System

Authors: Sivasundaram Karnan
Sivasundaram KarnanAffiliation: Department of Biochemistry, Aichi Medical University School of Medicine, Nagakute, Aichi, Japan
Bio-protocol author page: a3860
Akinobu Ota
Akinobu OtaAffiliation: Department of Biochemistry, Aichi Medical University School of Medicine, Nagakute, Aichi, Japan
Bio-protocol author page: a3861
Yuko Konishi
Yuko KonishiAffiliation: Department of Biochemistry, Aichi Medical University School of Medicine, Nagakute, Aichi, Japan
Bio-protocol author page: a3862
Md Wahiduzzaman
Md WahiduzzamanAffiliation: Department of Biochemistry, Aichi Medical University School of Medicine, Nagakute, Aichi, Japan
Bio-protocol author page: a3863
Shinobu Tsuzuki
Shinobu TsuzukiAffiliation: Department of Biochemistry, Aichi Medical University School of Medicine, Nagakute, Aichi, Japan
Bio-protocol author page: a3864
Yoshitaka Hosokawa
Yoshitaka HosokawaAffiliation: Department of Biochemistry, Aichi Medical University School of Medicine, Nagakute, Aichi, Japan
Bio-protocol author page: a3865
 and Hiroyuki Konishi
Hiroyuki KonishiAffiliation: Department of Biochemistry, Aichi Medical University School of Medicine, Nagakute, Aichi, Japan
For correspondence: hkonishi@aichi-med-u.ac.jp
Bio-protocol author page: a3866
date: 12/20/2016, 160 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2058.

[Abstract] Adeno-associated virus (AAV)-based targeting vectors have 1-4-log higher gene targeting efficiencies compared with plasmid-based targeting vectors. The efficiency of AAV-mediated gene targeting is further increased by introducing a promoter-trap system into targeting vectors. In addition, we found that the use of ribosome-skipping 2A peptide rather ...

Affinity Pulldown of Biotinylated RNA for Detection of Protein-RNA Complexes

Authors: Amaresh C Panda
Amaresh C PandaAffiliation: Laboratory of Genetics and Genomics, National Institute on Aging, National Institutes of Health, 251 Bayview Blvd., Baltimore, United States
For correspondence: amaresh.panda@nih.gov
Bio-protocol author page: a3875
Jennifer L. Martindale
Jennifer L. Martindale Affiliation: Laboratory of Genetics and Genomics, National Institute on Aging, National Institutes of Health, 251 Bayview Blvd., Baltimore, United States
Bio-protocol author page: a3880
 and Myriam Gorospe
Myriam GorospeAffiliation: Laboratory of Genetics and Genomics, National Institute on Aging, National Institutes of Health, 251 Bayview Blvd., Baltimore, United States
Bio-protocol author page: a3881
date: 12/20/2016, 228 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2062.

[Abstract] RNA-binding proteins (RBPs) have recently emerged as crucial players in the regulation of gene expression. The interactions of RBPs with target mRNAs control the levels of gene products by altering different regulatory steps, including pre-mRNA splicing and maturation, nuclear mRNA export, and mRNA stability and translation (Glisovic et al., 2008). ...

Gene Expression Analysis of Sorted Cells by RNA-seq in Drosophila Intestine

Authors: Jun Chen
Jun ChenAffiliation: National Institute of Biological Sciences, Beijing, China
Bio-protocol author page: a3929
Jia Li
Jia LiAffiliation: National Institute of Biological Sciences, Beijing, China
Bio-protocol author page: a16
Huaiwei Huang
Huaiwei HuangAffiliation: National Institute of Biological Sciences, Beijing, China
Bio-protocol author page: a3931
 and Rongwen Xi
Rongwen XiAffiliation: National Institute of Biological Sciences, Beijing, China
For correspondence: xirongwen@nibs.ac.cn
Bio-protocol author page: a3932
date: 12/20/2016, 185 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2079.

[Abstract] RNA sequencing (RNA-seq) has become a popular method for profiling gene expression. Among many applications, one common purpose is to identify differentially expressed genes and pathways in different biological or pathological conditions. This protocol provides detailed procedure for RNA-seq analysis of ~250,000 sorted Drosophila intestinal cells (Chen ...

Single-step Marker Switching in Schizosaccharomyces pombe Using a Lithium Acetate Transformation Protocol

Authors: Simon David Brown
Simon David BrownAffiliation: Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK
Bio-protocol author page: a3916
 and Alexander Lorenz
Alexander LorenzAffiliation: Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK
For correspondence: a.lorenz@abdn.ac.uk
Bio-protocol author page: a3917
date: 12/20/2016, 316 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2075.

[Abstract] The ability to utilize different selectable markers for tagging or mutating multiple genes in Schizosaccharomyces pombe is hampered by the historical use of only two selectable markers, ura4+ and kanMX6; the latter conferring resistance to the antibiotic G418 (geneticin). More markers have been described recently, but introducing these into yeast cells ...

Murine Leukemia Virus (MLV)-based Coronavirus Spike-pseudotyped Particle Production and Infection

Authors: Jean Kaoru Millet
Jean Kaoru MilletAffiliation: Department of Microbiology and Immunology, Cornell University, Ithaca NY, United States
For correspondence: jkm248@cornell.edu
Bio-protocol author page: a3793
 and Gary R. Whittaker
Gary R. WhittakerAffiliation: Department of Microbiology and Immunology, Cornell University, Ithaca NY, United States
For correspondence: gary.whittaker@cornell.edu
Bio-protocol author page: a942
date: 12/5/2016, 214 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2035.

[Abstract] Viral pseudotyped particles (pp) are enveloped virus particles, typically derived from retroviruses or rhabdoviruses, that harbor heterologous envelope glycoproteins on their surface and a genome lacking essential genes. These synthetic viral particles are safer surrogates of native viruses and acquire the tropism and host entry pathway characteristics ...

A Golden Gate-based Protocol for Assembly of Multiplexed gRNA Expression Arrays for CRISPR/Cas9

Authors: Johan Vad-Nielsen*
Johan Vad-NielsenAffiliation: Department of Biomedicine, Aarhus University, Aarhus C, Denmark
For correspondence: johanvn@biomed.au.dk
Bio-protocol author page: a3868
Lin Lin *
Lin Lin Affiliation: Department of Biomedicine, Aarhus University, Aarhus C, Denmark
For correspondence: lin.lin@biomed.au.dk
Bio-protocol author page: a3869
Kristopher Torp Jensen
Kristopher Torp JensenAffiliation: Department of Biomedicine, Aarhus University, Aarhus C, Denmark
Bio-protocol author page: a3870
Anders Lade Nielsen
Anders Lade Nielsen Affiliation: Department of Biomedicine, Aarhus University, Aarhus C, Denmark
Bio-protocol author page: a3871
 and Yonglun Luo
Yonglun LuoAffiliation: Department of Biomedicine, Aarhus University, Aarhus C, Denmark
Bio-protocol author page: a3872
 (*contributed equally to this work) date: 12/5/2016, 277 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2059.

[Abstract] The CRISPR (clustered regularly interspaced short palindromic repeats)-associated protein 9 (Cas9) has become the most broadly used and powerful tool for genome editing. Many applications of CRISPR-Cas9 require the delivery of multiple small guide RNAs (gRNAs) into the same cell in order to achieve multiplexed gene editing or regulation. Using traditional ...

In vitro Autophosphorylation and Phosphotransfer Assay of Cyanobacterial Histidine Kinase 2

Author: Iskander M. Ibrahim
Iskander M. IbrahimAffiliation: Faculty of Engineering and Science, University of Greenwich, Chatham Maritime, Kent, ME4 4TB, UK
For correspondence: I.M.Ibrahim@greenwich.ac.uk
Bio-protocol author page: a3795
date: 12/5/2016, 227 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2036.

[Abstract] This is a detailed protocol of an autophosphorylation and phosphotransfer activities of Synechocystis sp. PCC 6803 full-length Histidine Kinase 2 (Hik2) protein described by Ibrahim et al., 2016. In this protocol, radioactively labelled ATP was used to study an autophosphorylation and phosphotransfer activity of the full-length Hik2 protein. ...
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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, 79911 views, 30 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, 72739 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, 32887 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, 29587 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, 26028 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, 25737 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] Standard DNA Cloning

Author: Fanglian He date: 4/5/2011, 22830 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] 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, 22728 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] Lentivirus Production

Author: Nabila Aboulaich date: 3/5/2011, 21964 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, 20654 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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