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Tetracycline as Regulator of Inducible Gene Expression Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3986

Category: Transfection Protocols: Stable Transfection Protocols

Protocol uses an autoregulatory system in which the transcriptional trans-activator tTA drives its own expression and that of a target gene. The first stage of the procedure describes how to generate stable lines of NIH-3T3 cells that express either tTA alone or tTA and the tetracycline-regulated target gene. - [Read Tetracycline as Regulator of Inducible Gene Expression Protocol]

Tetracycline as Regulator of Inducible Gene Expression Protocol II - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3957

Category: Transfection Protocols: Stable Transfection Protocols

This stage of the procedure describes the transfection with target genes of cell lines already expressing inducible tTA. In this example, the target genes are transfected on a plasmid that carries puromycin resistance as a selectable marker. - [Read Tetracycline as Regulator of Inducible Gene Expression Protocol II]

Total RNA isolation (Guanidinium Thiocyanate-Phenol-Chloroform Extraction) Protocol - http://www.flemingtonlab.com/Protocols/TotalRNAprep.pdf

Category: Nucleic Acid Purification Protocols: RNA Isolation Protocols

Protocol for total RNA isolation. This method is fast and works well for preparation of total RNA. We typically use total RNA for quantitative S1 nuclease analysis of endogenous (or in vitro) gene expression, and this method works well for this purpose. - [Read Total RNA isolation (Guanidinium Thiocyanate-Phenol-Chloroform Extraction) Protocol]

Transfection/Retroviral Infection pCL-Eco+Gene of Interest into 293T Cells Infecting HC11 Cells - http://www.bcm.edu/rosenlab/protocols/retroviral.pdf

Category: Cloning Protocols: Vector Protocols

Protocol for the transfection/Retroviral Infection pCL-Eco+Gene of interest into 293T cells infecting HC11 cells. Includes: Split 293T cells for transfection; Transfect 293T cells; Split HC11 cells for infection; Infect target (HC11) cells. - [Read Transfection/Retroviral Infection pCL-Eco+Gene of Interest into 293T Cells Infecting HC11 Cells]

Transformation of Magnaporthe grisea to Phosphinothricin Resistance Protocol - http://www.fgsc.net/fgn42/leung.html

Category: Fungi Protocols: Fungi Genetics Protocols

Transformation of Magnaporthe grisea to phosphinothricin resistance using the bar gene from Streptomyces hygroscopicus. - [Read Transformation of Magnaporthe grisea to Phosphinothricin Resistance Protocol]

Transgene Expression in Regenerated Roots - http://www.cshprotocols.org/cgi/content/abstract/2007/3/pdb.prot4691

Category: Plant Biology Protocols: Plant Genetics Protocols

This procedure, which uses a root transformation protocol, provides a rapid method for assessing gene expression in Arabidopsis roots. It is useful for testing promoter:reporter gene constructs, for expressing genes, the overexpression of which is lethal in whole plants, and for transforming the roots of plants that are recalcitrant to conventional transformation techniques. The protocol has been used successfully with Ws, No-0, and RLD ecotypes. - [Read Transgene Expression in Regenerated Roots]

Transient Expression in Protoplasts - http://www.cshprotocols.org/cgi/content/abstract/2007/3/pdb.prot4692

Category: Plant Biology Protocols: Plant Genetics Protocols

The study of transient gene expression provides a useful complement to the study of stably transformed plants. Transient assays offer a quick method of testing the effects of genes, using either phenotypic, molecular, or biochemical readouts. Transient assays based on Agrobacterium-mediated transformation of leaf explants have been described for other plant species, but it is not known how well these assays work in Arabidopsis. - [Read Transient Expression in Protoplasts]

Transient Transfection Into 293T Cells Protocol - http://www.cbrinstitute.org/labs/springer/protocols/jun_293T.html

Category: Transfection Protocols: Transient Transfection Protocols

Transient transfection into 293T cells is a convenient way to overexpress and obtain both cellular and extracellular (secreted or membrane) proteins. 293 is a human renal epithelial cell line which is transformed by adenovirus E1A gene product. 293T is a derivative which also express SV40 large T antigen, allowing episomal replication of plasmids containing the SV40 origin and early promoter region. They (both) have the unusual property of being highly transfectable. - [Read Transient Transfection Into 293T Cells Protocol]

Two-hybrid Systems Stage 1: Characterization of a Bait-LexA Fusion Protein Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3895

Category: Yeast Protocols: Yeast Two-Hybrid Systems Protocols

Protocol describes how to generate a plasmid construct (pBAIT) that expresses a target protein fused to the bacterial LexA protein. PBAIT is cotransformed into yeast with a lexAop-lacZ reporter plasmid carrying the bacterial lacZ gene under the control of the lexA operator. The recipient yeast strain contains a chromosomally integrated leu2 reporter gene, also under the control of the lexA operator. - [Read Two-hybrid Systems Stage 1: Characterization of a Bait-LexA Fusion Protein Protocol]

Western Blot Analysis of Endogenous Gene Expression Protocol - http://www.flemingtonlab.com/Protocols/WestBlotAnalyofEndogProts.pdf

Category: Western Blot Protocols

Procedure describes how it denatures most of the modification and degradation proteins immediately giving the most accurate read out of the true levels of protein at the time of harvest. However, in cases where detection is a problem, a limited purification (e.g. isolation of nuclear extract for the detection of transcription factors) might be required to allow analysis. - [Read Western Blot Analysis of Endogenous Gene Expression Protocol]

Yeast Immunofluoresence Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4167

Category: Yeast Protocols: Yeast Staining Protocols

This protocol describes a method based on that of Pringle for immunofluorescent staining of yeast, as would be done in gene replacement experiments. - [Read Yeast Immunofluoresence 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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