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(LCM): Preparation and Sectioning of Frozen Tissue Blocks and Purification of RNA from Isolated Cel - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4107

Category: Primary Cell Isolation and Culture Protocols: Laser Capture Microdissection Protocols: Tissue Preparation for LCM Protocols

LCM isolates specific cells or tissues from samples mounted on microscope slides. The samples are viewed through a thermoplastic film that is attached to a microcentrifuge tube lid. Localized heat, caused by the application of a laser pulse, fuses the membrane to the cells of interest, which can then be harvested for further analysis. RNA and proteins can be purified from the isolated cells, allowing detailed analysis of gene expression. This protocol is divided into three stages. - [Read (LCM): Preparation and Sectioning of Frozen Tissue Blocks and Purification of RNA from Isolated Cel]

3' Rapid Amplification of cDNA Ends (RACE) Protocol - http://www.nottingham.ac.uk/~mbzspd/methods/3RACE_PCR.html

Category: RNA Protocols: 3' RACE Rapid Amplification of cDNA Ends Protocols

PCR cycle steps, protocol using SuperScript II reverse transcriptase (Life Technologies) and Taq polymerase, Gene specific primer, T17 Adapter primer and an Adapter primer. Dr.Dawson, Nottingham. - [Read 3' Rapid Amplification of cDNA Ends (RACE) Protocol]

3'-End cDNA Amplification Using Classic RACE Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/14/pdb.prot4130

Category: PCR Protocols: cDNA Synthesis Protocols

To generate "3'-end" partial cDNA clones, mRNA is reverse-transcribed using a "hybrid" primer (Qtotal, QT) that consists of two mixed bases (GATC/GAC followed by [T]17) and a unique 35-base oligonucleotide sequence (QI-QO). Amplification is then performed using a primer containing part of this sequence (Qouter, Qo) (which now binds to each cDNA at its 3'-end) and a primer derived from the gene of interest, GSP1 (gene-specific primer 1). - [Read 3'-End cDNA Amplification Using Classic RACE Protocol]

5'-End cDNA Amplification Using Classic RACE Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/14/pdb.prot4131

Category: PCR Protocols: cDNA Synthesis Protocols

To generate "5'-end" partial cDNA clones using classic RACE, the first-strand products are generated by reverse transcription (primer extension) from a known gene-specific primer (GSP-RT). Then, a poly(A) tail is appended using terminal deoxynucleotidyltransferase (Tdt) and dATP. Amplification is carried out using three primers. - [Read 5'-End cDNA Amplification Using Classic RACE Protocol]

A Rapid Method for Generating Gene Deletions in Aspergillus fumigatus Protocol - http://www.aspergillus.org.uk/indexhome.htm?secure/laboratory_protocols/./genedel.html~main

Category: Fungi Protocols: Aspergillus Protocols

The technique makes use of an Escherichia coli strain expressing the redΑßΓ operon under the control of an inducible promoter. This enables the strain to carry out homologous recombination with only 50-60 bp of homologous sequence. The procedure does not require any DNA ligation and is very rapid. It allows a single gene or region on a cosmid to be replaced by a bi-functional selectable marker (having both an E. coli and an A. fumigatus marker). - [Read A Rapid Method for Generating Gene Deletions in Aspergillus fumigatus Protocol]

Agrobacterium-Mediated Gene Transfer via Hypocotyls Protocol - http://wheat.pw.usda.gov/~lazo/methods/lazo/cottontr.html

Category: Plant Biology Protocols: Plant Genetics Protocols

Protocol describing Agrobacterium mediated gene transfer via hypocotyls. - [Read Agrobacterium-Mediated Gene Transfer via Hypocotyls Protocol]

Amplification of cDNA Generated by Reverse Transcription of mRNA Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3837

Category: PCR Protocols: cDNA Synthesis Protocols

An oligodeoxynucleotide primer hybridized to mRNA is extended by an RNA-dependent DNA polymerase to create a cDNA copy that can be amplified by PCR. Depending on the purpose of the experiment, the primer for first-strand cDNA synthesis can be specifically designed to hybridize to a particular target gene, or a general primer such as oligo(dT) can be used to prime cDNA synthesis from essentially all mammalian mRNAs - [Read Amplification of cDNA Generated by Reverse Transcription of mRNA Protocol]

Analysis of the Plant Epidermis Experiments - http://www.arabidopsis.org/cshl-course/6-epidermis.html

Category: Plant Biology Protocols: Arabidopsis Tissue Culture

Experiments included for analysis of plant epidermis: Steroid induction of gene expression in Arabidopsis; Replica Molds and Casts of the plant epidermis. - [Read Analysis of the Plant Epidermis Experiments]

Anatomy of a Comparative Gene Expression Study - http://www.cs.wustl.edu/~jbuhler//research/array/

Category: Genetics and Genomics: Cancer Genetics Protocols

Anatomy of a comparative gene expression study. Includes: Choosing Cell Populations; mRNA Extraction and Reverse Transcription; Fluorescent Labeling of cDNA's; Hybridization to a DNA Microarray; Scanning the Hybridized Array; Interpreting the Scanned Image. - [Read Anatomy of a Comparative Gene Expression Study]

Anatomy of a Comparative Gene Expression Study - http://www.cs.wustl.edu/~jbuhler//research/array/

Category: Microarray Protocols: Microarray Analysis Protocols

Information on anatomy of a comparative gene expression study. Includes: Choosing cell populations; mRNA Extraction and Reverse Transcription; Fluorescent labeling of cDNA's; Hybridization of DNA microarray; Scanning the hybridized array; Interpreting the scanned image. - [Read Anatomy of a Comparative Gene Expression Study]

Annealing, Cloning, and Sequencing of shRNA Linkers into pEN_H1 Plasmids Protocol - http://www.signaling-gateway.org/data/cgi-bin/ProtocolFile.cgi/afcs_PP00000230.pdf?pid=PP00000230

Category: RNAi Technology Protocols

Protocol describes the preparation of an shRNA-expressing Gateway entry vector. This process is preceded by the design and synthesis of target gene-specific sense and antisense oligonucleotides which, when annealed, will constitute an shRNA cassette. - [Read Annealing, Cloning, and Sequencing of shRNA Linkers into pEN_H1 Plasmids Protocol]

Antisense Oligonucleotide Transfection of RAW 264.7 Cells with FuGENE 6 in a 24-Well Dish - http://www.signaling-gateway.org/data/cgi-bin/ProtocolFile.cgi/afcs_PP00000261.pdf?pid=PP00000261

Category: Transfection Protocols: Stable Transfection Protocols

The AfCS is utilizing antisense technology to manipulate signaling protein expression in the RAW 264.7 macrophage-like cell line. This can be achieved by the transfection of gene-specific antisense oligonucleotides (ASOs). The following procedure involves the transfection of ASOs into RAW 264.7 cells using FuGENE 6 transfection reagent. Subsequently, the isolated total RNA or protein from these transfected cells can be used to assess the level of mRNA or protein knockdown, respectively. - [Read Antisense Oligonucleotide Transfection of RAW 264.7 Cells with FuGENE 6 in a 24-Well Dish]

Aspergillus nidulans Mutants - http://www.fgsc.net/alist.html

Category: Fungi Protocols: Aspergillus Protocols

Compilation of A. nidulans mutants listing names, affected enzymes (or gene functions), linkage assignments, properties of the mutants, and names of corresponding loci in N. crassa. - [Read Aspergillus nidulans Mutants]

Assay for Phage Containing the Beta-galactosidase Gene - http://humgen.wustl.edu/hdk_lab_manual/lambda/lambda7.html

Category: Reporter Gene Assays: B-Galactosidase Assay Protocols

This assay is used when working with phage vectors carrying the beta-gal gene. If the cloning event disrupts a normally functional copy of the gene in the vector the resulting plaques would appear clear in the assay. If the phages contain a functional beta-gal gene they will form blue rings around their plaques. Any strain which is not an overproducer of beta-gal will work as indicator host bacteria; a single chromosomal copy of the gene is not a problem. - [Read Assay for Phage Containing the Beta-galactosidase Gene]

Assay for Phage Containing the Beta-galactosidase Gene Protocol - http://humgen.wustl.edu/hdk_lab_manual/lambda/lambda7.html

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

This assay is used when working with phage vectors carrying the beta-galactosidase gene (often used for immunological screening). If the cloning event disrupts a normally functional copy of the gene in the vector the resulting plaques would appear clear in the assay. If the phages contain a functional beta-galactosidase gene they will form blue rings around their plaques. Any strain which is not an overproducer of beta- galactosidase will work as indicator host bacteria. - [Read Assay for Phage Containing the Beta-galactosidase Gene Protocol]

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

Category: Reporter Gene Assays: B-Galactosidase Assay Protocols

Protocol for Beta-galactosidase reporter gene assay (liquid form). - [Read Beta-galactosidase Reporter Gene Assay (Liquid Form)]

Bioresponsive Targeted Charge Neutral Lipid Vesicles for Systemic Gene Delivery Protocol - http://www.cshprotocols.org/cgi/content/short/2006/1/pdb.prot4450

Category: Gene Delivery Protocols: DNA Nanoparticles Protocols

This protocol describes a stepwise procedure to prepare nucleic acids encapsulated in a polyethylene glycol (PEG)-shielded nanolipoparticle (NLP) that contain a bioresponsive lipid and ligand. This process provides several advantages for systemic gene delivery. The in vivo circulation time is extended. Also, low pH-sensitive lipids enhance DNA unpacking and endosomal escape. Finally, ligands inserted into the NLP surface can target gene delivery to specific tissues or cells in vivo. - [Read Bioresponsive Targeted Charge Neutral Lipid Vesicles for Systemic Gene Delivery Protocol]

C. elegans Gene Knockout Protocol - http://www.zoology.ubc.ca/~alorch/koprotocol.htm

Category: C. elegans Protocols: C. elegans Genetics Protocols

The procedure is to mutagenize a large population of worms with trimethylpsoralen and UV irradiation, set up 1152 subpopulations, screen DNA made from this library for deletions in specific genes by nested PCR, and then to recover single worms carrying the deletions through a sib-selection process. - [Read C. elegans Gene Knockout Protocol]

Collection of Early Mouse Embryos for RNAi Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/21/pdb.prot4510

Category: RNAi Technology Protocols: RNAi Mouse Embryos and Oocytes Protocols

Protocol describes a method to collect early embryos from 6-week-old mice. Subsequently, the isolated embryos can be injected with double-stranded RNA to induce knockdown of a gene of interest. - [Read Collection of Early Mouse Embryos for RNAi Protocol]

Collection of Mouse Oocytes for RNAi Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/21/pdb.prot4509

Category: RNAi Technology Protocols: RNAi Mouse Embryos and Oocytes Protocols

Protocol describes a method to collect oocytes from 6-week-old mice. Subsequently, the isolated oocytes can be injected with double-stranded RNA to induce knockdown of a gene of interest. - [Read Collection of Mouse Oocytes for RNAi Protocol]

Colorimetric Assay to Identify Putative Ribofuranosylaminobenzene 5'-Phosphate Synthase Genes - http://76.162.25.80/bpo/arts/1/48/m48.pdf

Category: E Coli Protocols: E coli Genetics Protocols

Protocol for Colorimetric Assay to Identify Putative Ribofuranosylaminobenzene 5'-Phosphate Synthase Genes. The production of active RFAP synthase from Methanothermobacter thermautotrophicus was achieved by coexpression of the gene MTH0830 with a molecular chaperone. This is the first direct biochemical identification of a methanogen gene that codes for an active RFAP synthase. - [Read Colorimetric Assay to Identify Putative Ribofuranosylaminobenzene 5'-Phosphate Synthase Genes]

Competent Cell Preparation [CaCl2] - http://www.cf.ac.uk/biosi/staff/kille/Methods/Gene/Comp_cell_CaCl.html

Category: Microbiology: Bacterial Competent Cells Preparation E.Coli

Nice protocol for the preparation of Bacterial cells to allow uptake of plasmid vectors. Dr Peter Kille. Hot Metals Cardiff. - [Read Competent Cell Preparation [CaCl2]]

Conditional Ablation of Stat3 Socs3 Discloses the Dual Role for Reactive Astrocytes - http://www.natureprotocols.com/2006/06/30/conditional_ablation_of_stat3s.php

Category: Neuroscience Protocols: Neuronal Cell Culture Protocols: Rat Neuronal Cell Culture Protocols

Conditional ablation of stat3/socs3 discloses the dual role for reactive astrocytes after spinal cord injury. The current protocol demonstrates that reactive astrocytes play a crucial role in wound healing and functional recovery by using mice with a selective deletion of the signal transducer and activator of transcription-3 (STAT3) or suppression of cytokine signaling-3 (SOCS3) under the control of Nestin gene promoter/enhancer (STAT3N–/–, SOCS3N–/–). - [Read Conditional Ablation of Stat3 Socs3 Discloses the Dual Role for Reactive Astrocytes]

Cosuppression in C. elegans Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4318

Category: C. elegans Protocols: C. elegans Genetics Protocols

The cosuppression effect in C. elegans does not spread throughout the animal. Cosuppression in C. elegans can be triggered by highly repetitive transgenes that contain gene constructs. - [Read Cosuppression in C. elegans Protocol]

Cosuppression in C. elegans Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4318

Category: RNAi Technology Protocols: RNAi C. elegans Protocols

Cosuppression is a process in Caenorhabditis elegans that closely resembles RNAi. In contrast to RNAi, however, the cosuppression effect in C. elegans does not spread throughout the animal. Cosuppression in C. elegans can be triggered by highly repetitive transgenes that contain gene constructs. - [Read Cosuppression in C. elegans 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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