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How to Create Gene Knockouts in Neurospora - http://www.fgsc.net/neurosporaprotocols/How%20to%20create%20gene%20knockouts%20in%20Neurospora.pdf

Category: Fungi Protocols: Neurospora Protocols

Information and tips on how to create gene knockouts in Neurospora. - [Read How to Create Gene Knockouts in Neurospora]

How to Determine Gene Order Using 3-Point Crosses - http://www.fgsc.net/neurosporaprotocols/How%20to%20determine%20gene%20order%20using%203-point%20crosses%20final.pdf

Category: Fungi Protocols: Neurospora Protocols

Information and tips on how to determine gene order using 3-point crosses. - [Read How to Determine Gene Order Using 3-Point Crosses]

How to Determine Whether a Gene is Essential for Survival - http://www.fgsc.net/neurosporaprotocols/How%20to%20determine%20whether%20a%20gene%20is%20essential%20final.pdf

Category: Fungi Protocols: Neurospora Protocols

Information and tips on how to determine whether a gene is essential for survival. - [Read How to Determine Whether a Gene is Essential for Survival]

How to Use the mtr Gene as a Selectable Marker for Loss or Gain of Function - http://www.fgsc.net/neurosporaprotocols/mtr.pdf

Category: Fungi Protocols: Neurospora Protocols

Information and tips on how to use the mtr gene as a selectable marker for loss or gain of function. - [Read How to Use the mtr Gene as a Selectable Marker for Loss or Gain of Function]

Hydrodynamics-Based Gene Transduction Protocol - http://www.natureprotocols.com/2006/06/29/hydrodynamicsbased_gene_transd.php

Category: Gene Delivery Protocols

This highly efficient in vivo gene transduction technique for laboratory mice. Hepatocytes are most effectively transduced by tail vein injection of a large volume of DNA solution in a short time. Practice with the injection technique is necessary!!! - [Read Hydrodynamics-Based Gene Transduction Protocol]

Immunofluorescence Detection of RNAi-Induced Protein Knockdown in Mammalian Cells Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4344

Category: Immunology: Immunofluorescence Protocols

This protocol describes the use of a specific antibody that recognizes the targeted gene product to detect RNAi-induced gene knockdown in mammalian cells. Western blot technology can be used as an alternative (see Detection of RNAi-Induced Protein Knockdown in Mammalian Cells by Western Blotting). - [Read Immunofluorescence Detection of RNAi-Induced Protein Knockdown in Mammalian Cells Protocol]

Immunofluorescence Detection of RNAi-Induced Protein Knockdown in Mammalian Cells Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4344

Category: RNAi Technology Protocols: RNAi Mammalian Cells Protocols

Protocol describes the use of a specific antibody that recognizes the targeted gene product to detect RNAi-induced gene knockdown in mammalian cells. Western blot technology can be used as an alternative. - [Read Immunofluorescence Detection of RNAi-Induced Protein Knockdown in Mammalian Cells Protocol]

Immunofluorescent Staining for the Laser Microdissection of Individual Cells Protocol - http://www.natureprotocols.com/2006/08/03/immunofluorescent_staining_for.php

Category: Primary Cell Isolation and Culture Protocols: Laser Capture Microdissection Protocols

Useful techniques to circumvent disruption of tissue structure in the analysis of gene expression are LCM and LDM. While they require specialized microscopes and systems, they are similar in that freshly-cut frozen tissue sections can be microdissected using either a general histological stain (like H&E) or by staining with fluorescently conjugated antibodies. The LCM system by Arcturus involves... - [Read Immunofluorescent Staining for the Laser Microdissection of Individual Cells Protocol]

In Vivo Functional Real-Time Imaging of Transplanted Islets Using Positron Emission Tomography (PET) - http://www.natureprotocols.com/2006/12/21/seeing_is_believing_in_vivo_fu.php

Category: Microscopy Protocols: In Vivo Imaging Protocols

Positron emission tomography (PET) is a established quantitative and noninvasive imaging modality. With the PET reporter gene (PRG)/PET reporter probe (PRP) system, based on a mutant form of herpes simplex virus 1 thymidine kinase (HSV1-sr39tk), the PET signal is directly proportional to the enzymatic activity of sr39TK9-14. In this protocol, we describe in detail a method for reporter gene labeling of islets and quantitative scanning using a reporter probe. - [Read In Vivo Functional Real-Time Imaging of Transplanted Islets Using Positron Emission Tomography (PET)]

Isolation of Arabidopsis Nuclei and Measurement of Gene Transcription Rates Protocol - http://www.natureprotocols.com/2007/01/25/isolation_of_arabidopsis_nucle.php

Category: Plant Biology Protocols: Arabidopsis Genetics Protocols

Protocol for the isolation of Arabidopsis nuclei and measurement of gene transcription rates using nuclear run-on assays. Plant materials are ground in hexylene glycol-based buffers and highly enriched nuclear fractions are obtained using Percoll density gradients. Standard and small-scale protocols are presented, along with a tested method for nuclear run-on assays. The entire process may be completed within 3 days. - [Read Isolation of Arabidopsis Nuclei and Measurement of Gene Transcription Rates Protocol]

Kanamycin Selection of Transformed Arabidopsis Protocol - http://www.cshprotocols.org/cgi/content/abstract/2006/30/pdb.prot4669

Category: Plant Biology Protocols: Arabidopsis Tissue Culture

The most commonly used markers for selection of transgenic Arabidopsis are resistance to the antibiotic kanamycin and to the herbicide glufosinate ammonium. Resistance to kanamycin is conferred by a bacterial gene encoding the enzyme neomycin phosphotransferase (NPT). In this protocol, kanamycin-resistant seedlings are selected on solid medium. - [Read Kanamycin Selection of Transformed Arabidopsis Protocol]

Lipoplex and LPD Nanoparticles for In Vivo Gene Delivery Protocol - http://www.cshprotocols.org/cgi/content/abstract/2006/1/pdb.prot4448

Category: Gene Delivery Protocols: DNA Nanoparticles Protocols

Lipoplex (cationic liposome-DNA complex) is formed via electrostatic interaction of anionic nucleic acids with cationic liposomes. A thin film of lipids is dried on the bottom of a glass tube and rehydrated in an aqueous solution. The resulting liposome suspension is passed through polycarbonate filters of desired pore size. This protocol also describes the preparation, physical properties, and biological activity of liposome-polycation-DNA (LPD) nanoparticles. - [Read Lipoplex and LPD Nanoparticles for In Vivo Gene Delivery Protocol]

Liquid Beta-galactosidase Reporter Gene Assay Protocol - http://www.med.unc.edu/~hdohlman/bgal.html

Category: Yeast Protocols: Yeast Genetics Protocols: B-Galactosidase Assay Protocols

Protocol for liquid beta-galactosidase reporter gene assay. - [Read Liquid Beta-galactosidase Reporter Gene Assay Protocol]

Methods for Analyzing the Role of DNA Methylation and Chromatin Structure in Regulating T Lymphocyte - http://www.biologicalprocedures.com/bpo/arts/1/89/m89.pdf

Category: Nucleic Acid Modification Protocols: DNA Methylation Protocols

Methods for analyzing the role of DNA methylation and chromatin structure in regulating T lymphocyte gene expression. - [Read Methods for Analyzing the Role of DNA Methylation and Chromatin Structure in Regulating T Lymphocyte]

Microarray Analysis of Highly Purified Human Decidual NK Cells Protocol - http://www.natureprotocols.com/2006/08/07/microarray_analysis_of_highly.php

Category: Microarray Protocols: Microarray Analysis Protocols

Description of gene array analysis and normalization - [Read Microarray Analysis of Highly Purified Human Decidual NK Cells Protocol]

Microdissection Overview - http://cgap-mf.nih.gov/Protocols/Microdissection/Overview.html

Category: Primary Cell Isolation and Culture Protocols: Laser Capture Microdissection Protocols

Human tissues are comprised of multiple interacting cell populations in a complex three dimensional arrangement with each cellular phenotype determined by a unique profile of mRNA and protein expression. Before microdissection techniques were developed, the only analysis tools for phenotypic studies were primarily immunohistochemistry and in-situ hybridization. While useful, these tools are limited to single gene analysis and, in general, do not allow qualitative studies. - [Read Microdissection Overview]

Microinjection of dsRNA into Drosophila Embryos Protocol - http://www.cshprotocols.org/cgi/content/full/2006/21/pdb.prot4516

Category: Microinjection Protocols: Microinjection of DNA Protocols

This protocol provides a description of how to introduce double-stranded RNA (dsRNA) into Drosophila embryos by microinjection. Several days of preparation are required before injections into Drosophila embryos begin. Flies must be in abundant supply for egg collection. Bombardment of embryos with dsRNA-coated gold particles (Delivery of dsRNA into Drosophila Embryos by a Gene Gun) can be used as an alternative. - [Read Microinjection of dsRNA into Drosophila Embryos Protocol]

Molecular and Genetic Toxicology Studies on Gene Mutation in Mammalian Cells In Vitro - http://www.sri.com/biosciences/bdd/toxic/protocol.html#5

Category: Pharmacology and Toxicology Protocols: Cytotoxicity Protocols

Molecular and genetic toxicology studies on gene mutation in mammalian cells In vitro. Includes: G201 Mouse Lymphoma L5178Y/tk+/Cell Gene Mutation Assay; G201-R; G202 Mouse Lymphoma L5178Y/tk+/Cell Gene Mutation Screening Assay; G203 Chinese Hamster Ovary or V79 Cell Mutation Assay at the hprt Locus; G203R. - [Read Molecular and Genetic Toxicology Studies on Gene Mutation in Mammalian Cells In Vitro]

Monster Green® Fluorescent Protein Assay - http://www.promega.com/catalog/country_select.asp?/tbs/tb320/tb320.html&ckt=2

Category: Reporter Gene Assays: GFP Assay Protocols

Green fluorescent protein is commonly used to monitor gene expression and protein trafficking within intact cells. The Monster Green® Fluorescent Protein is encoded by an improved synthetic version of the green fluorescent protein gene originally cloned from Montastrea cavernosa (Great Star Coral). - [Read Monster Green® Fluorescent Protein Assay]

Multiplex Real-Time PCR Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4113

Category: PCR Protocols: Real-Time PCR Protocols

In multiplex real-time PCR, different sets of primers with different labels are used to amplify separate genes from the template DNA in one tube. This protocol uses LUX (Light Upon eXtension) primers from invitrogen. FAM (6-carboxy-fluorescein) is used to label the gene of interest, and JOE (6-carboxy-4', 5'-dichloro-2',7'-dimethoxy-fluorescein) is used to label a housekeeping gene as an internal control to normalize between different reactions. - [Read Multiplex Real-Time PCR Protocol]

Nucleosomal Array Disruption Assay Protocol - http://www.bio.com/protocolstools/protocol.jhtml%3Bjsessionid%3D3APITJJIWFIM1R3FQLMCFEWHUWBNSIV0?id=p9058

Category: Drosophila Protocols: Drosophila Genetics Protocols

Protocol describes how to test whether a transcription factor disrupts the chromatin of a promoter of a gene of interest. First, chromatin is assembled in vitro on the gene of interest in the presence and absence of a transcriptional activator (see Protocol on Assembly of Chromatin with Drosophila S-190 Chromatin Assembly Extract and Transcriptional Activators). - [Read Nucleosomal Array Disruption Assay Protocol]

PCR-Mediated Gene Disruption: One-Step Method Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4169

Category: PCR Protocols

DNA prepared by PCR-mediated gene disruption can be used to transform yeast in gene replacement experiments. This protocol uses two primers, tailed with approximately 50 nucleotides homologous to the gene of interest, that target insertion of the PCR product to that locus. Each primer ends with a universal sequence that is designed to amplify various selectable markers from plasmid templates. - [Read PCR-Mediated Gene Disruption: One-Step Method Protocol]

PEI Nanoparticles for Targeted Gene Delivery Protocol - http://www.cshprotocols.org/cgi/content/abstract/2006/1/pdb.prot4451

Category: Gene Delivery Protocols: DNA Nanoparticles Protocols

This protocol describes the preparation of polyethylenimine (PEI)/DNA nanoparticles for targeted gene delivery. This delivery strategy improves the efficiency of gene transfer by enhancing the entry of gene vectors into the desired cells and reducing uptake by nontarget cells. We describe here methods for the conjugation of targeting peptides to PEIs, formation of DNA complexes using the conjugated PEIs or nonconjugated PEIs together with targeting peptides, and cell transfection. - [Read PEI Nanoparticles for Targeted Gene Delivery Protocol]

Placenta Specific Gene Manipulation by Transducing Zona-Free Blastocyst using Lentiviral Vector - http://www.natureprotocols.com/2007/01/24/placenta_specific_gene_manipul.php

Category: Cytogenetics Protocols

Details a placenta specific gene manipulation by transducing blastocysts with lentiviral vectors1. After a removal of zona pellucida which functions as a physical barrier, trophoblast cells lying outermost layer of blastocyst were transduced from outside with high-titer lentiviral vectors. As most placental cells descend from trophoblast cells while fetus originated from inner cell mass, transgene expression can be observed in trophoblast cells from preimplantation stages and in placenta... - [Read Placenta Specific Gene Manipulation by Transducing Zona-Free Blastocyst using Lentiviral Vector]

Precision Engineering of Plant Gene Loci by Homologous Recombination Cloning in E coli Protocol - http://www.plantmethods.com/content/1/1/6

Category: E Coli Protocols: E coli Genetics Protocols

Protocol for precision engineering of plant gene loci by homologous recombination cloning in Escherichia coli. Describe the basis for homologous recombination cloning in E. coli, the available tools and resources, together with a protocol for long range cloning and manipulation of an Arabidopsis thaliana gene locus, to create constructs co-ordinately driven by locus-specific regulatory elements. - [Read Precision Engineering of Plant Gene Loci by Homologous Recombination Cloning in E coli Protocol]

Articles (1-10 of 15)

Populational Analysis of Genomic DNA Protocol

A protocol on the populational Analysis of genomic DNA.

Genomic DNA Labeling of Microarrays Protocol

DNA microarrays are an ordered arrangement of DNA molecules complementary to genes of interest that are "spotted" by robotic equipment onto a glass slide substrate. The expression of genes in cells can be monitored with microarrays by preparing cDNA from the mRNA of cells of interest and measuring the hybridization to the microarray. This protocol describes the labeling of genomic DNA for use as a probe for hybridization to the cDNA spotted on the array.

Tubulin Polymerization Protocol using GTP and GMPCPP

Tubulin is polymerized into microtubules by incubating tubulin at 37°C with GTP. A nucleation seed is added when the purpose is to assay microtubule elongation. Tubulin can also be polymerized for the purposes of recycling the tubulin or labeling the microtubules with fluorescently labeled tubulin. Based on the protocol by Timothy Mitchison of Harvard University.

Microarray Protocol Preparation of Fluorescent DNA Probes from Human mRNA

This Microarray Protocol Preparation of Fluorescent DNA Probes from Human mRNA protocol describes the production of probes labeled with the fluorescent dyes, Cy3 and Cy5, following the synthesis of cDNA from human mRNA and the hybridization of the probes to DNA microarrays.

Purifying DNA Fragments from Agarose Gels Alternate Protocol

An alternate protocol for purifying DNA fragments from agarose gels.

Constructing Random-Peptide Libraries for Phage Display

The protocol describes the construction of a large (10^8 to 10^10) primary random peptide phage library in fUSE5 or f88-4 strains.

3' Rapid Amplification of cDNA Ends RACE Using PCR Protocol

3' Rapid Amplification of cDNA Ends RACE Using PCR Protocol. This protocol contains the steps for 3' end rapid amplification of mRNA by PCR. The first-strand cDNA is synthesized from total or poly(A+) RNA by priming from the poly-A tail of the mRNA using a oligo (dT) adaptor primer. The cDNA is then amplified via PCR using a gene-specific primer and an adaptor primer.

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