promoter Protocols

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]

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]

Category: Yeast Protocols: Yeast Protein Protocols

This assay is performed to detect ubiquitylated proteins in yeast. Yeast that have been transformed with a vector expressing polyhistidine-tagged ubiquitin (Ub) under the control of a copper-inducible promoter are grown, induced with copper, and harvested. Total ubiquitylated proteins are then recovered by nickel-affinity chromatography, and specific proteins are detected by Western blotting. - [Read Detection of Ubiquitylated Proteins in Yeast Protocol]

Category: Viral Manipulation Protocols: Phage Lamda Manipulation Protocols

Protocol describes how (1) to clone cloned sequences encoding open reading frames in plasmids carrying bacteriophage {lambda} pL promoters, (2) to optimize expression of target proteins in transformants carrying these recombinants, and (3) to scale-up production of foreign proteins. - [Read Expression of Cloned Genes in E. coli Using the Bacteriophage lambda pL Promoter Protocol]

Category: E Coli Protocols: E coli Protein Protocols

Protocol for expression of cloned genes in E. coli using the bacteriophage lambda pL promoter. Protocol describes how (1) to clone cloned sequences encoding open reading frames in plasmids carrying bacteriophage lambda pL promoters, (2) to optimize expression of target proteins in transformants carrying these recombinants, and (3) to scale-up production of foreign proteins. - [Read Expression of Cloned Genes in E. coli Using the Bacteriophage lambda pL Promoter Protocol]

Category: E Coli Protocols: E coli Protein Protocols

Protocol for the expression of cloned genes in E. coli using the bacteriophage T7 promoter. Protocol describes how (1) to clone cloned sequences encoding open reading frames in plasmids carrying bacteriophage T7 promoters, (2) to optimize expression of target proteins in transformants carrying these recombinants, and (3) to scale-up production of foreign proteins. - [Read Expression of Cloned Genes in E. coli Using the Bacteriophage T7 Promoter Protocol]

Category: Microbiology: Bacterial E.Coli Culture Media & Plates

How to prepare M9 minimal media and grow in M9 media. Chazin Lab , Vanderbilt Univ. - [Read Expression Protocol in M9 Minimal Media via T7 Promoter]

Category: Bacterial Cell Culture Protocols: Bacterial Cell Culture Media Protocols

This protocol has been used successfully to 15N or 13C/15N label our proteins using our pET1120/BL21(DE3) expression system: Preparing M9 minimal media begins with preparing a 5x stock solution of M9 salts. Generally, M9 salts contain a nitrogen source in the form of NH4Cl. Since we want to add a labeled nitrogen source, our 5x salts are prepared minus NH4Cl. Standard 5 X M9 Minimal Media salts minus nitrogen source For 1L 5xM9 salts: - [Read Expression Protocol in M9 Minimal Media via T7 Promoter]

Category: DNA Microarray Protocols: DNA Methylation Microarray CpG

Kimura et al., NAR 2005. "easy-handling technology provided reproducible and precise measurement of methylated CpGs in MGMT promoter and, thus, our method may bring about a potential evolution in the handling of a variety of high-throughput DNA methylatio - [Read Methylation profiles of genes utilizing newly developed CpG island methylation microarray on colorec]

Category: Cell Biology and Cell Culture: In Vitro Reconstitution Protocols

Protocol describes a system which includes all of the necessary components for in vitro transcription as well as a positive control template that provides run-off transcripts from a CMV immediate early promoter. This system is designed for runoff transcription. Alternatively, transcription products can be analyzed by primer extension. - [Read Nuclear Extract in vitro Transcription System]

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]

Category: Avian Bird Protocols

Protocol describes the production of double-stranded RNA (dsRNA) from fragments of cDNAs of candidate genes. The cDNA fragments must be cloned in plasmids with a flanking SP6 and T7 promoter (e.g., pSP72 or pCRII). The plasmid is linearized and sense and antisense RNAs are produced separately by in vitro transcription. After purification, the RNA strands are annealed to yield a dsRNA molecule suitable for RNAi in avian embryos. - [Read Production of dsRNA for RNAi in Avian Embryos Protocol]

Category: RNAi Technology Protocols: RNAi Avian Embryos Protocols

Protocol describes the production of double-stranded RNA (dsRNA) from fragments of cDNAs of candidate genes.  The cDNA fragments must be cloned in plasmids with a flanking SP6 and T7 promoter (e.g., pSP72 or pCRII).  The plasmid is linearized and sense and antisense RNAs are produced separately by in vitro transcription. - [Read Production of dsRNA for RNAi in Avian Embryos Protocol]

Category: Signalling Signal Transduction Protocols

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–/–). Procedure includes: Surgical procedures, Functional evaluation, Immunohistochemistry, In vitro migration assay. - [Read Protocol for Conditional Ablation of stat3/socs3 Discloses the Dual Role for Reactive Astrocytes]

Category: RNA Protocols: In Vitro Transcription T7 SP6 T3 Protocols

Ribomax T7 In Vitro Transcription Protocol. Synthesis of mg quantities of RNA from a T7 promoter. Promega. - [Read Ribomax T7 In Vitro Transcription Protocol]

Category: Cytogenetics Protocols: RNA In Situ Hybridization Protocols

Protocol for RNA labeling by in vitro transcription of DNA with DIG, Biotin or Fluorescein RNA Labeling Mix. A PCR fragment that has the appropriate promoter ligated to its 5’-ends can also serve as a transcription template. The procedure described incorporates one modified nucleotide (DIG-, Biotin-, or Fluorescein-UTP) at approximately every 20 – 25th position in the transcripts. - [Read RNA Labeling by In Vitro Transcription of DNA with DIG, Biotin or Fluorescein RNA Labeling Mix]

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]

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]

Category: Yeast Protocols: Yeast Two-Hybrid Systems Protocols

In this stage of the protocol, a mammalian cDNA library constructed in a plasmid such as pJG4-5 is transformed into yeast strains containing pBAIT and the lexAop-lacZ reporter plasmid. PJG4-5 expresses the cloned cDNAs from a cassette containing a transcriptional activation domain and other moieties under the control of the yeast GAL1 promoter. - [Read Two-hybrid Systems Stage 2: Selecting an Interactor Protocol]