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Molecular Biology

Gene Dosage Experiments in Enterobacteriaceae Using Arabinose-regulated Promoters

Featured protocol,  Authors: Sanchari Bhattacharyya
Sanchari BhattacharyyaAffiliation: Department of Chemistry and Chemical Biology, Harvard University, Cambridge MA, USA
Bio-protocol author page: a4838
Shimon Bershtein
Shimon BershteinAffiliation: Department of Life Sciences, Ben-Gurion University of the Negev, POB 653, Beer-Sheva, Israel
Bio-protocol author page: a4839
 and Eugene I Shakhnovich
Eugene I ShakhnovichAffiliation: Department of Chemistry and Chemical Biology, Harvard University, Cambridge MA, USA
For correspondence: shakhnovich@chemistry.harvard.edu
Bio-protocol author page: a4840
date: 7/20/2017, 14 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2396.

Brief version appeared in Elife, Dec 2016
This protocol is used to assay the effect of protein over-expression on fitness of E. coli. It is based on a plasmid expression of a protein of interest from an arabinose-regulated pBAD promoter followed by the measurement of the intracellular protein abundance by Western blot along with the measurement of growth parameters of E. coli cell expressing this protein.

GUS Staining of Guard Cells to Identify Localised Guard Cell Gene Expression

Featured protocol,  Authors: Zhao Liu
Zhao LiuAffiliation: Hebei Key Laboratory of Molecular and Cellular Biology, Key Laboratory of Molecular and Cellular Biology of Ministry of Education, Hebei Collaboration Innovation Center for Cell Signaling, College of Life Science, Hebei Normal University, Shijiazhuang, China
Bio-protocol author page: a4858
Wei Wang
Wei WangAffiliation: Basic Forestry and Proteomics Research Center, Haixia Institute of Science and Technology, Fujian Agriculture and Forestry University, Fuzhou, China
Bio-protocol author page: a4859
Chun-Guang Zhang
Chun-Guang ZhangAffiliation: Hebei Key Laboratory of Molecular and Cellular Biology, Key Laboratory of Molecular and Cellular Biology of Ministry of Education, Hebei Collaboration Innovation Center for Cell Signaling, College of Life Science, Hebei Normal University, Shijiazhuang, China
Bio-protocol author page: a4860
Jun-Feng Zhao
Jun-Feng ZhaoAffiliation: Hebei Key Laboratory of Molecular and Cellular Biology, Key Laboratory of Molecular and Cellular Biology of Ministry of Education, Hebei Collaboration Innovation Center for Cell Signaling, College of Life Science, Hebei Normal University, Shijiazhuang, China
Bio-protocol author page: a4861
 and Yu-Ling Chen
Yu-Ling Chen Affiliation: Hebei Key Laboratory of Molecular and Cellular Biology, Key Laboratory of Molecular and Cellular Biology of Ministry of Education, Hebei Collaboration Innovation Center for Cell Signaling, College of Life Science, Hebei Normal University, Shijiazhuang, China
For correspondence: yulingchen@mail.hebtu.edu.cn
Bio-protocol author page: a4862
date: 7/20/2017, 12 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2446.

Brief version appeared in Plant Physiol, Jun 2017
Determination of a gene expression in guard cells is essential for studying stomatal movements. GUS staining is one means of detecting the localization of a gene expression in guard cells. If a gene is specially expressed in guard cells, the whole cotyledons or rosette leaf can be used for GUS staining. However, if a gene is expressed in both mesophyll and guard cells, it is hard to exhibit a clear expression of the gene in guard cells by a GUS staining image from leaf. To gain a clear guard cell GUS image of small G protein ROP7, a gene expressed in both mesophyll and guard cells, we peeled the epidermal strips from the leaf of 3-4 week-old plants. After removing the mesophyll cells, the epidermal strips were used for GUS staining. We compared the GUS staining images from epidermal strips or leaf of small G protein ROP7 and RopGEF4, a gene specifically expressed in guard cells, and found that GUS staining of epidermal strips provided a good method to show the guard cell expression of a gene expressed in both mesophyll and guard cells. This protocol is applicable for any genes that are expressed in guard cells of Arabidopsis, or other plants that epidermal strips can be easily peeled from the leaf.

Generation of Targeted Knockout Mutants in Arabidopsis thaliana Using CRISPR/Cas9

Featured protocol,  Authors: Florian Hahn
Florian HahnAffiliation: Institute of Plant Biochemistry, Cluster of Excellence on Plant Science (CEPLAS), Center for Synthetic Life Sciences (CSL), Heinrich Heine University, Düsseldorf, Germany
Bio-protocol author page: a4806
Marion Eisenhut
Marion EisenhutAffiliation: Institute of Plant Biochemistry, Cluster of Excellence on Plant Science (CEPLAS), Center for Synthetic Life Sciences (CSL), Heinrich Heine University, Düsseldorf, Germany
Bio-protocol author page: a4807
Otho Mantegazza
Otho MantegazzaAffiliation: Institute of Plant Biochemistry, Cluster of Excellence on Plant Science (CEPLAS), Center for Synthetic Life Sciences (CSL), Heinrich Heine University, Düsseldorf, Germany
Bio-protocol author page: a4808
 and Andreas P. M. Weber
Andreas P. M. WeberAffiliation: Institute of Plant Biochemistry, Cluster of Excellence on Plant Science (CEPLAS), Center for Synthetic Life Sciences (CSL), Heinrich Heine University, Düsseldorf, Germany
For correspondence: Andreas.Weber@uni-duesseldorf.de
Bio-protocol author page: a4809
date: 7/5/2017, 224 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2384.

Brief version appeared in Front Plant Sci, Jan 2017
The CRISPR/Cas9 system has emerged as a powerful tool for gene editing in plants and beyond. We have developed a plant vector system for targeted Cas9-dependent mutagenesis of genes in up to two different target sites in Arabidopsis thaliana. This protocol describes a simple 1-week cloning procedure for a single T-DNA vector containing the genes for Cas9 and sgRNAs, as well as the detection of induced mutations in planta. The procedure can likely be adapted for other transformable plant species.

Multiplex Gene Editing via CRISPR/Cas9 System in Sheep

Featured protocol,  Authors: Yiyuan Niu
Yiyuan NiuAffiliation: College of Animal Science and Technology, Northwest A&F University, Yangling, China
Bio-protocol author page: a4803
Yi Ding
Yi DingAffiliation: College of Animal Science and Technology, Northwest A&F University, Yangling, China
Bio-protocol author page: a4804
Xiaolong Wang
Xiaolong WangAffiliation: College of Animal Science and Technology, Northwest A&F University, Yangling, China
Bio-protocol author page: a4805
 and Yulin Chen
Yulin ChenAffiliation: College of Animal Science and Technology, Northwest A&F University, Yangling, China
For correspondence: chenyulin@nwafu.edu.cn
Bio-protocol author page: a4802
date: 7/5/2017, 168 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2385.

Brief version appeared in Sci Rep, Aug 2016
Sheep is a major large animal model for studying development and disease in biomedical research. We utilized CRISPR/Cas9 system successfully to modify multiple genes in sheep. Here we provide a detailed protocol for one-cell-stage embryo manipulation by co-injecting Cas9 mRNA and RNA guides targeting three genes (MSTN, ASIP, and BCO2) to create genetic-modified sheep. Procedure described sgRNA design, construction of gRNA-Cas9 plasmid, efficient detection in fibroblast, embryos and sheep, and some manipulative technologies. Our findings suggested that the CRISPR/Cas9 method can be exploited as a powerful tool for livestock improvement by targeting multiple genes that are in charge of economically significant traits simultaneously.

Gene Dosage Experiments in Enterobacteriaceae Using Arabinose-regulated Promoters

Authors: Sanchari Bhattacharyya
Sanchari BhattacharyyaAffiliation: Department of Chemistry and Chemical Biology, Harvard University, Cambridge MA, USA
Bio-protocol author page: a4838
Shimon Bershtein
Shimon BershteinAffiliation: Department of Life Sciences, Ben-Gurion University of the Negev, POB 653, Beer-Sheva, Israel
Bio-protocol author page: a4839
 and Eugene I Shakhnovich
Eugene I ShakhnovichAffiliation: Department of Chemistry and Chemical Biology, Harvard University, Cambridge MA, USA
For correspondence: shakhnovich@chemistry.harvard.edu
Bio-protocol author page: a4840
date: 7/20/2017, 14 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2396.

[Abstract] This protocol is used to assay the effect of protein over-expression on fitness of E. coli. It is based on a plasmid expression of a protein of interest from an arabinose-regulated pBAD promoter followed by the measurement of the intracellular protein abundance by Western blot along with the measurement of growth parameters of E. coli cell expressing ...

GUS Staining of Guard Cells to Identify Localised Guard Cell Gene Expression

Authors: Zhao Liu
Zhao LiuAffiliation: Hebei Key Laboratory of Molecular and Cellular Biology, Key Laboratory of Molecular and Cellular Biology of Ministry of Education, Hebei Collaboration Innovation Center for Cell Signaling, College of Life Science, Hebei Normal University, Shijiazhuang, China
Bio-protocol author page: a4858
Wei Wang
Wei WangAffiliation: Basic Forestry and Proteomics Research Center, Haixia Institute of Science and Technology, Fujian Agriculture and Forestry University, Fuzhou, China
Bio-protocol author page: a4859
Chun-Guang Zhang
Chun-Guang ZhangAffiliation: Hebei Key Laboratory of Molecular and Cellular Biology, Key Laboratory of Molecular and Cellular Biology of Ministry of Education, Hebei Collaboration Innovation Center for Cell Signaling, College of Life Science, Hebei Normal University, Shijiazhuang, China
Bio-protocol author page: a4860
Jun-Feng Zhao
Jun-Feng ZhaoAffiliation: Hebei Key Laboratory of Molecular and Cellular Biology, Key Laboratory of Molecular and Cellular Biology of Ministry of Education, Hebei Collaboration Innovation Center for Cell Signaling, College of Life Science, Hebei Normal University, Shijiazhuang, China
Bio-protocol author page: a4861
 and Yu-Ling Chen
Yu-Ling Chen Affiliation: Hebei Key Laboratory of Molecular and Cellular Biology, Key Laboratory of Molecular and Cellular Biology of Ministry of Education, Hebei Collaboration Innovation Center for Cell Signaling, College of Life Science, Hebei Normal University, Shijiazhuang, China
For correspondence: yulingchen@mail.hebtu.edu.cn
Bio-protocol author page: a4862
date: 7/20/2017, 12 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2446.

[Abstract] Determination of a gene expression in guard cells is essential for studying stomatal movements. GUS staining is one means of detecting the localization of a gene expression in guard cells. If a gene is specially expressed in guard cells, the whole cotyledons or rosette leaf can be used for GUS staining. However, if a gene is expressed in both mesophyll ...

Generation of Targeted Knockout Mutants in Arabidopsis thaliana Using CRISPR/Cas9

Authors: Florian Hahn
Florian HahnAffiliation: Institute of Plant Biochemistry, Cluster of Excellence on Plant Science (CEPLAS), Center for Synthetic Life Sciences (CSL), Heinrich Heine University, Düsseldorf, Germany
Bio-protocol author page: a4806
Marion Eisenhut
Marion EisenhutAffiliation: Institute of Plant Biochemistry, Cluster of Excellence on Plant Science (CEPLAS), Center for Synthetic Life Sciences (CSL), Heinrich Heine University, Düsseldorf, Germany
Bio-protocol author page: a4807
Otho Mantegazza
Otho MantegazzaAffiliation: Institute of Plant Biochemistry, Cluster of Excellence on Plant Science (CEPLAS), Center for Synthetic Life Sciences (CSL), Heinrich Heine University, Düsseldorf, Germany
Bio-protocol author page: a4808
 and Andreas P. M. Weber
Andreas P. M. WeberAffiliation: Institute of Plant Biochemistry, Cluster of Excellence on Plant Science (CEPLAS), Center for Synthetic Life Sciences (CSL), Heinrich Heine University, Düsseldorf, Germany
For correspondence: Andreas.Weber@uni-duesseldorf.de
Bio-protocol author page: a4809
date: 7/5/2017, 224 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2384.

[Abstract] The CRISPR/Cas9 system has emerged as a powerful tool for gene editing in plants and beyond. We have developed a plant vector system for targeted Cas9-dependent mutagenesis of genes in up to two different target sites in Arabidopsis thaliana. This protocol describes a simple 1-week cloning procedure for a single T-DNA vector containing the genes for ...

Multiplex Gene Editing via CRISPR/Cas9 System in Sheep

Authors: Yiyuan Niu
Yiyuan NiuAffiliation: College of Animal Science and Technology, Northwest A&F University, Yangling, China
Bio-protocol author page: a4803
Yi Ding
Yi DingAffiliation: College of Animal Science and Technology, Northwest A&F University, Yangling, China
Bio-protocol author page: a4804
Xiaolong Wang
Xiaolong WangAffiliation: College of Animal Science and Technology, Northwest A&F University, Yangling, China
Bio-protocol author page: a4805
 and Yulin Chen
Yulin ChenAffiliation: College of Animal Science and Technology, Northwest A&F University, Yangling, China
For correspondence: chenyulin@nwafu.edu.cn
Bio-protocol author page: a4802
date: 7/5/2017, 168 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2385.

[Abstract] Sheep is a major large animal model for studying development and disease in biomedical research. We utilized CRISPR/Cas9 system successfully to modify multiple genes in sheep. Here we provide a detailed protocol for one-cell-stage embryo manipulation by co-injecting Cas9 mRNA and RNA guides targeting three genes (MSTN, ASIP, and BCO2) to create genetic-modified ...

RNA Capping by Transcription Initiation with Non-canonical Initiating Nucleotides (NCINs): Determination of Relative Efficiencies of Transcription Initiation with NCINs and NTPs

Authors: Jeremy G. Bird
Jeremy G. BirdAffiliation 1: Department of Genetics and Waksman Institute, Rutgers University, Piscataway, USA
Affiliation 2: Department of Chemistry and Waksman Institute, Rutgers University, Piscataway, USA
Bio-protocol author page: a4666
Bryce E. Nickels
Bryce E. NickelsAffiliation: Department of Genetics and Waksman Institute, Rutgers University, Piscataway, USA
For correspondence: bnickels@waksman.rutgers.edu
Bio-protocol author page: a4667
 and Richard H. Ebright
Richard H. EbrightAffiliation: Department of Chemistry and Waksman Institute, Rutgers University, Piscataway, USA
For correspondence: ebright@waksman.rutgers.edu
Bio-protocol author page: a4668
date: 6/20/2017, 238 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2336.

[Abstract] It recently has been established that adenine-containing cofactors, including nicotinamide adenine dinucleotide (NAD+), reduced nicotinamide adenine dinucleotide (NADH), and 3’-desphospho-coenzyme A (dpCoA), can serve as ‘non-canonical initiating nucleotides’ (NCINs) for transcription initiation by bacterial and eukaryotic cellular RNA polymerases ...

Dense sgRNA Library Construction Using a Molecular Chipper Approach

Authors: Jijun Cheng
Jijun ChengAffiliation 1: Department of Genetics, Yale University School of Medicine, New Haven, Connecticut, USA
Affiliation 2: Yale Stem Cell Center, Yale Cancer Center, Yale University School of Medicine, New Haven, Connecticut, USA
For correspondence: j.cheng@yale.edu
Bio-protocol author page: a4740
Wen Pan
Wen PanAffiliation 1: Department of Genetics, Yale University School of Medicine, New Haven, Connecticut, USA
Affiliation 2: Yale Stem Cell Center, Yale Cancer Center, Yale University School of Medicine, New Haven, Connecticut, USA
Bio-protocol author page: a3289
 and Jun Lu
Jun LuAffiliation 1: Department of Genetics, Yale University School of Medicine, New Haven, Connecticut, USA
Affiliation 2: Yale Stem Cell Center, Yale Cancer Center, Yale University School of Medicine, New Haven, Connecticut, USA
Affiliation 3: Yale Cooperative Center of Excellence in Hematology, Yale University, New Haven, Connecticut, USA
Affiliation 4: Yale Center for RNA Science and Medicine, Yale University, New Haven, Connecticut, USA
For correspondence: jun.lu@yale.edu
Bio-protocol author page: a4741
date: 6/20/2017, 258 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2373.

[Abstract] Genetic screens using single-guide-RNA (sgRNA) libraries and CRISPR technology have been powerful to identify genetic regulators for both coding and noncoding regions of the genome. Interrogating functional elements in noncoding regions requires sgRNA libraries that are densely covering, and ideally inexpensive, easy to implement and flexible for customization. ...

In vitro Assay to Measure DNA Polymerase β Nucleotide Insertion Coupled with the DNA Ligation Reaction during Base Excision Repair

Authors: Melike Çağlayan
Melike ÇağlayanAffiliation: Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, USA
Bio-protocol author page: a1849
 and Samuel H. Wilson
Samuel H. WilsonAffiliation: Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, USA
For correspondence: wilson5@niehs.nih.gov
Bio-protocol author page: a1850
date: 6/20/2017, 278 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2341.

[Abstract] We previously reported that oxidized nucleotide insertion by DNA polymerase β (pol β) can confound the DNA ligation step during base excision repair (BER) (Çağlayan et al., 2017). Here, we describe a method to investigate pol β nucleotide insertion coupled with DNA ligation, in the same reaction mixture including dGTP or 8-oxo-dGTP, pol β and DNA ligase ...

Single-molecule RNA Fluorescence in situ Hybridization (smFISH) in Caenorhabditis elegans

Authors: ChangHwan Lee
ChangHwan LeeAffiliation 1: Howard Hughes Medical Institute, University of Wisconsin-Madison, Madison, Wisconsin, 53706, USA
Affiliation 2: Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA
Bio-protocol author page: a4726
Hannah S. Seidel
Hannah S. SeidelAffiliation 1: Howard Hughes Medical Institute, University of Wisconsin-Madison, Madison, Wisconsin, 53706, USA
Affiliation 2: Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA
Present address: Department of Biology, Eastern Michigan University, Ypsilanti, Michigan, USA
Bio-protocol author page: a4727
Tina R. Lynch
Tina R. LynchAffiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA
Bio-protocol author page: a4728
Erika B. Sorensen
Erika B. SorensenAffiliation 1: Howard Hughes Medical Institute, University of Wisconsin-Madison, Madison, Wisconsin, 53706, USA
Affiliation 2: Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA
Present address: Department of Biology, Wabash College, Crawfordsville, Indiana, USA
Bio-protocol author page: a4729
Sarah L. Crittenden
Sarah L. CrittendenAffiliation: Howard Hughes Medical Institute, University of Wisconsin-Madison, Madison, Wisconsin, 53706, USA
Bio-protocol author page: a4730
 and Judith Kimble
Judith KimbleAffiliation 1: Howard Hughes Medical Institute, University of Wisconsin-Madison, Madison, Wisconsin, 53706, USA
Affiliation 2: Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA
For correspondence: jekimble@wisc.edu
Bio-protocol author page: a4731
date: 6/20/2017, 293 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2357.

[Abstract] Single-molecule RNA fluorescence in situ hybridization (smFISH) is a technique to visualize individual RNA molecules using multiple fluorescently-labeled oligonucleotide probes specific to the target RNA (Raj et al., 2008; Lee et al., 2016a). We adapted this technique to visualize RNAs in the C. elegans whole adult worm or its germline, which enabled ...

Modification of 3’ Terminal Ends of DNA and RNA Using DNA Polymerase θ Terminal Transferase Activity

Authors: Trung M. Hoang
Trung M. HoangAffiliation: Fels Institute for Cancer Research, Department of Medical Genetics and Molecular Biochemistry, Temple University Lewis Katz School of Medicine, Philadelphia, Pennsylvania, USA
Bio-protocol author page: a4648
Tatiana Kent
Tatiana KentAffiliation: Fels Institute for Cancer Research, Department of Medical Genetics and Molecular Biochemistry, Temple University Lewis Katz School of Medicine, Philadelphia, Pennsylvania, USA
Bio-protocol author page: a4649
 and Richard T. Pomerantz
Richard T. PomerantzAffiliation: Fels Institute for Cancer Research, Department of Medical Genetics and Molecular Biochemistry, Temple University Lewis Katz School of Medicine, Philadelphia, Pennsylvania, USA
For correspondence: richard.pomerantz@temple.edu
Bio-protocol author page: a4650
date: 6/20/2017, 205 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2330.

[Abstract] DNA polymerase θ (Polθ) is a promiscuous enzyme that is essential for the error-prone DNA double-strand break (DSB) repair pathway called alternative end-joining (alt-EJ). During this form of DSB repair, Polθ performs terminal transferase activity at the 3’ termini of resected DSBs via templated and non-templated nucleotide addition cycles. Since human ...

Single Genome Sequencing of Expressed and Proviral HIV-1 Envelope Glycoprotein 120 (gp120) and nef Genes

Authors: David J. Nolan
David J. NolanAffiliation 1: Bioinfoexperts, LLC, Thibodaux, Louisiana, USA
Affiliation 2: Department of Pathology, Immunology and Laboratory Medicine, Emerging Pathogens Institute, University of Florida, Gainesville, Florida, USA
Bio-protocol author page: a4654
Susanna L. Lamers
Susanna L. LamersAffiliation: Bioinfoexperts, LLC, Thibodaux, Louisiana, USA
Bio-protocol author page: a4655
Rebecca Rose
Rebecca RoseAffiliation: Bioinfoexperts, LLC, Thibodaux, Louisiana, USA
Bio-protocol author page: a4656
James J. Dollar
James J. DollarAffiliation 1: Bioinfoexperts, LLC, Thibodaux, Louisiana, USA
Affiliation 2: Department of Pathology, Immunology and Laboratory Medicine, Emerging Pathogens Institute, University of Florida, Gainesville, Florida, USA
Bio-protocol author page: a4657
Marco Salemi
Marco SalemiAffiliation: Department of Pathology, Immunology and Laboratory Medicine, Emerging Pathogens Institute, University of Florida, Gainesville, Florida, USA
Bio-protocol author page: a4658
 and Michael S. McGrath
Michael S. McGrathAffiliation 1: Departments of Laboratory Medicine, Pathology, and Medicine, University of California at San Francisco, San Francisco, California, USA
Affiliation 2: The AIDS and Cancer Specimen Resource, University of California at San Francisco, San Francisco, California, USA
For correspondence: MMcGrath@php.ucsf.edu
Bio-protocol author page: a4659
date: 6/20/2017, 215 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2334.

[Abstract] The current study provides detailed protocols utilized to amplify the complete HIV-1 gp120 and nef genes from single copies of expressed or integrated HIV present in fresh-frozen autopsy tissues of patients who died while on combined antiretroviral therapy (cART) with no detectable plasma viral load (pVL) at death (Lamers et al., 2016a and 2016b; Rose ...
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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, 93841 views, 32 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, 86877 views, 47 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, 37809 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, 33012 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, 28961 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, 28581 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, 26371 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, 26244 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, 24419 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, 24230 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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