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

Featured protocol,  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, 32 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2035.

Brief version appeared in PNAS, Oct 2014
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 governed by the heterologous envelope glycoprotein used. They have proven to be very useful tools used in research with many applications, such as enabling the study of entry pathways of enveloped viruses and to generate effective gene-delivery vectors. The basis for their generation lies in the capacity of some viruses, such as murine leukemia virus (MLV), to incorporate envelope glycoproteins of other viruses into a pseudotyped virus particle. These can be engineered to contain reporter genes such as luciferase, enabling quantification of virus entry events upon pseudotyped particle infection with susceptible cells. Here, we detail a protocol enabling generation of MLV-based pseudotyped particles, using the Middle East respiratory syndrome coronavirus (MERS-CoV) spike (S) as an example of a heterologous envelope glycoprotein to be incorporated. We also describe how these particles are used to infect susceptible cells and to perform a quantitative infectivity readout by a luciferase assay.

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

Featured protocol,  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, 34 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2059.

Brief version appeared in Cell Mol Life Sci, Nov 2016
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 co-transfection of single gRNA expression vectors, the likelihood of delivering several gRNAs into the same cell decreases in accordance with the number of gRNAs. Thus, we have developed a method to efficiently assemble gRNA expression cassettes (2-30 gRNAs) into one single vector using a Golden-Gate assembly method (Vad-Nielsen et al., 2016). In this protocol, we describe the detailed step-by-step instructions for assembly of the multiplexed gRNA expression array. The gRNA scaffold used in our expression array is the gRNA 1.0 system for the Cas9 protein from Streptococcus pyogenes driven by the human U6 promoter. 

In vitro Autophosphorylation and Phosphotransfer Assay of Cyanobacterial Histidine Kinase 2

Featured protocol,  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, 29 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2036.

Brief version appeared in Front Plant Sci, Feb 2016
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.

Biochemical Analysis of Caspase-8-dependent Proteolysis of IRF3 in Virus-infected Cells

Featured protocol,  Authors: Gayatri Subramanian
Gayatri SubramanianAffiliation: Department of Medical Microbiology and Immunology, University of Toledo College of Medicine and Life Sciences, Toledo, USA
Bio-protocol author page: a3735
Karen Pan
Karen PanAffiliation: Department of Medical Microbiology and Immunology, University of Toledo College of Medicine and Life Sciences, Toledo, USA
Bio-protocol author page: a3736
Ritu Chakravarti
Ritu ChakravartiAffiliation: Department of Surgery, University of Toledo College of Medicine and Life Sciences, Toledo, USA
Bio-protocol author page: a3737
 and Saurabh Chattopadhyay
Saurabh ChattopadhyayAffiliation: Department of Medical Microbiology and Immunology, University of Toledo College of Medicine and Life Sciences, Toledo, USA
For correspondence: Saurabh.Chattopadhyay@UToledo.edu
Bio-protocol author page: a3738
date: 11/20/2016, 145 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2018.

Brief version appeared in J Biol Chem, Sep 2011
Interferon regulatory factor 3 (IRF3) is a transcription factor, which is critical for the antiviral response against a wide range of viruses (Hiscott, 2007; Ikushima et al., 2013). It gets activated in virus-infected cells via Toll like receptors (TLRs), RIG-I (retinoic acid inducible gene 1) like receptors (RLRs), cyclic GMP-AMP synthase (cGAS) – stimulator of interferon genes (STING), which are sensors of viral components in the cells (Chattopadhyay and Sen, 2014a; 2014b; Hiscott, 2007). IRF3 is a cytoplasmic protein, upon activation by virally activated sensors it gets phosphorylated, translocated to the nucleus and binds to the interferon-sensitive response element (ISRE) of the gene promoters to induce their transcription (Hiscott, 2007). IRF3 has other functions, including direct stimulation of apoptosis in virus-infected cells. In this pathway, the transcriptional activity of IRF3 is not required (Chattopadhyay et al., 2013b; Chattopadhyay et al., 2016; Chattopadhyay et al., 2010; Chattopadhyay and Sen, 2010; Chattopadhyay et al., 2011). These pathways are negatively regulated by host factors as well as by viruses. Our studies indicate that IRF3 can be proteolytically processed by caspase-8-dependent cleavage (Sears et al., 2011). A specific site in IRF3 is targeted by caspase-8, activated in RNA or DNA virus-infected and dsRNA-stimulated cells (Sears et al., 2011). The direct involvement of caspase-8 was confirmed by in vitro cleavage assay using recombinant proteins and in vivo by virus activated caspase-8. The proteolytic cleavage of IRF3 can be inhibited by chemical inhibition or genetic ablation of caspase-8. The cleavage of IRF3 removes the activated pool of IRF3 and thus can be used as a pro-viral mechanism (Figure 1). Using a C-terminally epitope-tagged human IRF3, we analyzed the cleavage of IRF3 in virus-infected cells. Moreover, we used recombinant proteins in vitro to conclude that IRF3 is a substrate of caspase-8 (Sears et al., 2011). In the current protocol, we have outlined a simple and detailed procedure to biochemically analyze the proteolysis of IRF3 in virus-infected cells and the specific role of caspase-8 in this process.

Transformation of Thermus Species by Natural Competence

Featured protocol,  Authors: Alba Blesa
Alba BlesaAffiliation: Department of Molecular Biology, Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid - Consejo Superior de Investigaciones Científicas, Madrid, Spain
Bio-protocol author page: a3710
 and José Berenguer
José BerenguerAffiliation: Department of Molecular Biology, Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid - Consejo Superior de Investigaciones Científicas, Madrid, Spain
For correspondence: jberenguer@cbm.csic.es
Bio-protocol author page: a3711
date: 11/20/2016, 121 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2007.

Brief version appeared in J Bacteriol, Jan 2015
Many Thermus species harbour genomes scourged with horizontally transferred signatures. Thermus thermophilus (Tth) has been characterized as naturally competent. The transformation protocol described here is based on the maximum DNA uptake rates registered at mid-exponential phase (OD600 0.3-0.4). Here we describe the stepwise protocol followed for transformation of both plasmids and linearized genomic DNA, of which the latter can be employed as an alternative method to electroporation to introduce mutations or to generate gene deletions in Thermus isolates, for instance.

Cell-to-cell DNA Transfer among Thermus Species

Featured protocol,  Authors: Alba Blesa
Alba BlesaAffiliation: Department of Molecular Biology, Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid - Consejo Superior de Investigaciones Científicas, Madrid, Spain
Bio-protocol author page: a3710
 and José Berenguer
José BerenguerAffiliation: Department of Molecular Biology, Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid - Consejo Superior de Investigaciones Científicas, Madrid, Spain
For correspondence: jberenguer@cbm.csic.es
Bio-protocol author page: a3711
date: 11/20/2016, 109 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2006.

Brief version appeared in J Bacteriol, Jan 2015
The ability to transfer DNA via direct cell-to-cell contact-dependent process similar to conjugation has been described in Thermus thermophilus (Tth). Here, we detail the mating experiment protocol involving the lateral transfer of thermostable antibiotic resistance markers (i.e., kanamycin: KmR; hygromycin: HygR) between Thermus cells, enabling the selection and quantification of the transfer frequencies. Briefly, liquid cultures of both mates are mixed and laid onto a nitrocellulose filter on a TB plate. After incubation at 60 °C, filters are resuspended upon selective plating. The contribution of DNA uptake by transformation is abolished by the addition of DNase I to the mix. This protocol can be used for the transfer of large DNA fragments (> 10 kb) to Thermus species.

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, 32 views, 0 Q&A, How to cite
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, 34 views, 0 Q&A, How to cite
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, 29 views, 0 Q&A, How to cite
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.
Keywords: ...

Biochemical Analysis of Caspase-8-dependent Proteolysis of IRF3 in Virus-infected Cells

Authors: Gayatri Subramanian
Gayatri SubramanianAffiliation: Department of Medical Microbiology and Immunology, University of Toledo College of Medicine and Life Sciences, Toledo, USA
Bio-protocol author page: a3735
Karen Pan
Karen PanAffiliation: Department of Medical Microbiology and Immunology, University of Toledo College of Medicine and Life Sciences, Toledo, USA
Bio-protocol author page: a3736
Ritu Chakravarti
Ritu ChakravartiAffiliation: Department of Surgery, University of Toledo College of Medicine and Life Sciences, Toledo, USA
Bio-protocol author page: a3737
 and Saurabh Chattopadhyay
Saurabh ChattopadhyayAffiliation: Department of Medical Microbiology and Immunology, University of Toledo College of Medicine and Life Sciences, Toledo, USA
For correspondence: Saurabh.Chattopadhyay@UToledo.edu
Bio-protocol author page: a3738
date: 11/20/2016, 145 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2018.

[Abstract] Interferon regulatory factor 3 (IRF3) is a transcription factor, which is critical for the antiviral response against a wide range of viruses (Hiscott, 2007; Ikushima et al., 2013). It gets activated in virus-infected cells via Toll like receptors (TLRs), RIG-I (retinoic acid inducible gene 1) like receptors (RLRs), cyclic GMP-AMP synthase (cGAS) – ...

Transformation of Thermus Species by Natural Competence

Authors: Alba Blesa
Alba BlesaAffiliation: Department of Molecular Biology, Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid - Consejo Superior de Investigaciones Científicas, Madrid, Spain
Bio-protocol author page: a3710
 and José Berenguer
José BerenguerAffiliation: Department of Molecular Biology, Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid - Consejo Superior de Investigaciones Científicas, Madrid, Spain
For correspondence: jberenguer@cbm.csic.es
Bio-protocol author page: a3711
date: 11/20/2016, 121 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2007.

[Abstract] Many Thermus species harbour genomes scourged with horizontally transferred signatures. Thermus thermophilus (Tth) has been characterized as naturally competent. The transformation protocol described here is based on the maximum DNA uptake rates registered at mid-exponential phase (OD600 0.3-0.4). Here we describe the stepwise protocol followed for ...

Cell-to-cell DNA Transfer among Thermus Species

Authors: Alba Blesa
Alba BlesaAffiliation: Department of Molecular Biology, Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid - Consejo Superior de Investigaciones Científicas, Madrid, Spain
Bio-protocol author page: a3710
 and José Berenguer
José BerenguerAffiliation: Department of Molecular Biology, Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid - Consejo Superior de Investigaciones Científicas, Madrid, Spain
For correspondence: jberenguer@cbm.csic.es
Bio-protocol author page: a3711
date: 11/20/2016, 109 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2006.

[Abstract] The ability to transfer DNA via direct cell-to-cell contact-dependent process similar to conjugation has been described in Thermus thermophilus (Tth). Here, we detail the mating experiment protocol involving the lateral transfer of thermostable antibiotic resistance markers (i.e., kanamycin: KmR; hygromycin: HygR) between Thermus cells, enabling the ...

Reconstruction of the Mouse Inflammasome System in HEK293T Cells

Authors: Hexin Shi
Hexin ShiAffiliation: Center for the Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, Texas, USA
Bio-protocol author page: a3655
Anne Murray
Anne Murray Affiliation: Center for the Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, Texas, USA
Bio-protocol author page: a3656
 and Bruce Beutler
Bruce BeutlerAffiliation: Center for the Genetics of Host Defense, University of Texas Southwestern Medical Center, Dallas, Texas, USA
For correspondence: Bruce.Beutler@utsouthwestern.edu
Bio-protocol author page: a3657
date: 11/5/2016, 175 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.1986.

[Abstract] The NLRP3 (NLR family, Pyrin domain containing 3) inflammasome is a multiprotein complex comprised of NLRP3, pro-caspase-1, the adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC), and the protein kinase NIMA related kinase 7 (NEK7) (Shi et al., 2016; He et al., 2016; Schmid-Burgk et al., 2016). When cells are exposed to ...

Identification of Methylated Deoxyadenosines in Genomic DNA by dA6m DNA Immunoprecipitation

Authors: Magdalena J. Koziol
Magdalena J. KoziolAffiliation 1: Wellcome Trust Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge, UK
Affiliation 2: Department of Zoology, University of Cambridge, Cambridge, UK
For correspondence: mjk39@cam.ac.uk
Bio-protocol author page: a3666
Charles R. Bradshaw
Charles R. BradshawAffiliation: Wellcome Trust Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge, UK
Bio-protocol author page: a3667
George E. Allen
George E. AllenAffiliation: Wellcome Trust Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge, UK
Bio-protocol author page: a3668
Ana S. H. Costa
Ana S. H. CostaAffiliation: Medical Research Council Cancer Unit, University of Cambridge, Hutchison/MRC Research Centre, Cambridge, UK
Bio-protocol author page: a3669
 and Christian Frezza
Christian FrezzaAffiliation: Medical Research Council Cancer Unit, University of Cambridge, Hutchison/MRC Research Centre, Cambridge, UK
Bio-protocol author page: a3670
date: 11/5/2016, 179 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.1990.

[Abstract] dA6m DNA immunoprecipitation followed by deep sequencing (DIP-Seq) is a key tool in identifying and studying the genome-wide distribution of N6-methyldeoxyadenosine (dA6m). The precise function of this novel DNA modification remains to be fully elucidated, but it is known to be absent from transcriptional start sites and excluded from exons, suggesting ...

Sequencing of Ebola Virus Genomes Using Nanopore Technology

Author: Thomas Hoenen
Thomas HoenenAffiliation: Friedrich-Loeffler-Institut, Greifswald – Isle of Riems, Germany
For correspondence: thomas.hoenen@fli.de
Bio-protocol author page: a3685
date: 11/5/2016, 304 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.1998.

[Abstract] Sequencing of virus genomes during disease outbreaks can provide valuable information for diagnostics, epidemiology, and evaluation of potential countermeasures. However, particularly in remote areas logistical and technical challenges can be significant. Nanopore sequencing provides an alternative to classical Sanger and next-generation sequencing ...

Generation of Mitochondrial-nuclear eXchange Mice via Pronuclear Transfer

Authors: Robert A. Kesterson
Robert A. KestersonAffiliation: Department of Genetics, University of Alabama, Birmingham, USA
For correspondence: kesterso@uab.edu
Bio-protocol author page: a3629
Larry W. Johnson
Larry W. JohnsonAffiliation: Department of Genetics, University of Alabama, Birmingham, USA
Bio-protocol author page: a3630
Laura J. Lambert
Laura J. LambertAffiliation: Department of Genetics, University of Alabama, Birmingham, USA
Bio-protocol author page: a3631
Jay L. Vivian
Jay L. VivianAffiliation: Department of Pathology, University of Kansas Medical Center, Kansas City, USA
Bio-protocol author page: a3632
Danny R. Welch
Danny R. WelchAffiliation: Department of Cancer Biology, University of Kansas Cancer Center, Kansas City, USA
For correspondence: dwelch@kumc.edu
Bio-protocol author page: a3633
 and Scott W. Ballinger
Scott W. BallingerAffiliation: Division of Molecular and Cellular Pathology, University of Alabama, Birmingham, USA
For correspondence: sballing@uab.edu
Bio-protocol author page: a3634
date: 10/20/2016, 239 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.1976.

[Abstract] The mitochondrial paradigm for common disease proposes that mitochondrial DNA (mtDNA) sequence variation can contribute to disease susceptibility and progression. To test this concept, we developed the Mitochondrial-nuclear eXchange (MNX) model, in which isolated embryonic pronuclei from one strain of species are implanted into an enucleated embryo ...
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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, 77723 views, 30 Q&A, How to cite
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, 70628 views, 46 Q&A, How to cite
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, 32314 views, 7 Q&A, How to cite
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, 29068 views, 0 Q&A, How to cite
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, 25545 views, 2 Q&A, How to cite
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, 25319 views, 11 Q&A, How to cite
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, 22405 views, 4 Q&A, How to cite
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, 22245 views, 3 Q&A, How to cite
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, 21605 views, 6 Q&A, How to cite
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, 20290 views, 0 Q&A, How to cite
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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