interest Protocols

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]

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]

Category: PCR Protocols: cDNA Synthesis Protocols

New RACE, a variation of RNA ligase-mediated-RACE (RLM-RACE) (Liu and Gorovsky 1993) departs from classic RACE (see 5'-End cDNA Amplification Using Classic RACE) in that an "anchor" primer is attached to the 5'-end of the mRNA before the reverse transcription step; hence the anchor sequence becomes incorporated into the first-strand cDNA if, and only if, the reverse transcription proceeds through the entire length of the mRNA of interest. - [Read 5'-End cDNA Amplification Using New RACE Protocol]

Category: Cell Biology and Cell Culture

The protocol presented in this Application Sheet uses an alternative strategy to sedimentation on to a density barrier, that is to adjust the density of whole blood to a value just greater than the cells of interest and allow them to float to the surface. - [Read C8 Isolation of bovine peripheral blood mononuclear cells by flotation.]

Category: Immunology: Chemotaxis

Chemotaxis Assay, Springer Lab. A chemotaxis assay's function is to assess whether a factor or molecule of interest has chemotactic activity on a motile cell type. Chemotaxis is the ability of a factor to cause the migration of a cell. The chemotactic assay is based on the creation of a chemical gradient of the chemotactic agent which will cause cells to migrate through the gradient towards the chemotactic agent. - [Read Chemotaxis Assay]

Category: Cell Biology and Cell Culture

This chemotaxis assay protocol is based on the premise of creating a gradient of the chemotactic agent and allowing cells to migrate through a membrane towards the chemotactic agent. A chemotaxis assay can determine whether your protein or small molecule of interest has chemotactic activity on a specific cell type. Chemotaxis is then the ability of a protein to direct the migration of a specific cell. - [Read Chemotaxis Assay Protocol]

Category: Immunology: Immunoprecipitation Protocols: Chromatin Immunoprecipitation Protocols

Protocol describes the use of chromatin immunoprecipitation technology (ChIP) to analyze interactions of proteins or protein complexes with DNA in vivo. In this approach, the material is fixed with formaldehyde to preserve DNA-protein and protein-protein associations, the cells are lysed, and the chromatin is cut and solubilized. The chromatin suspension is immunoprecipitated with an antibody against the protein(s) of interest, and the coimmunoprecipitated DNA fragments are analyzed. - [Read Chromatin Immunoprecipitation (ChIP) of Protein Complexes Protocol]

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]

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]

Category: PCR Protocols: Reverse Transcriptase RT-PCR Protocols

The first step in competitive RT-PCR is the synthesis and purification of the synthetic competitor.  This is an RNA molecule designed to be reverse-transcribed and PCR-amplified with the same efficiency as the endogenous transcript of interest.  Once the competitor molecule has been prepared, as described in this protocol, competitive PCR can be carried out. - [Read Competitive RT-PCR: Preparation of Competitor RNA Protocol]

Category: Plant Biology Protocols: Plant Genetics Protocols

Virus-induced gene silencing (VIGS) uses a virus to deliver a sequence from a gene of interest into a host plant. The virus carrying the fragment of the gene of interest must be capable of replication if dsRNA is to be produced. One or two leaves are inoculated with Agrobacterium strains carrying the VIGS vector possessing the gene fragment. The virus then replicates and spreads throughout the plant, mediating silencing. - [Read Delivery of dsRNA into Plants by VIGS Methodology]

Category: DNA Protocols: Genomic DNA Library Protocols

COS-7 cells are transfected with a library of plasmids containing the genomic DNA of interest. - [Read Electroporation of the Library into COS-7 Cells for Exon Trapping and Amplification]

Category: Plant Biology Protocols: Arabidopsis Genetics Protocols

EMS is used at concentrations that induce multiple point mutations in each plant, such that mutant alleles of a specific locus are found at a rate of ~1 in 2000-5000 M2 plants. This high rate of mutagenesis makes possible the screening of relatively few plants to find those with the phenotype of interest, a particular advantage if the screen is laborious or if only a small number of genes mutate to the required phenotype. - [Read EMS Mutagenesis of Arabidopsis Seed Protocol]

Category: PCR Protocols: PCR Optimization

Method for amplifying DNA enzymatically by the polymerase chain reaction (PCR), including procedures to quickly determine conditions for successful amplification of the sequence and primer sets of interest, and to optimize for specificity, sensitivity, and yield.  The first step of PCR simply entails mixing template DNA, two appropriate oligonucleotide primers, Taq or other thermostable DNA polymerases, deoxyribonucleoside triphosphates (dNTPs), and a buffer. - [Read Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization Protocol]

Category: PCR Protocols

Protocol is the first in a set of three describing fluorescent mRNA differential display (FDD or FDDRT-PCR).  The method begins with the harvesting of total RNA from the tissue-cultured cells of interest.  For other starting materials, such as blood samples, please see Extraction and Purification of RNA from Blood Samples for Fluorescent mRNA Differential Display. - [Read Extraction and Purification of RNA from Tissue-Cultured Cells for Fluorescent mRNA Differential]

Category: DNA Protocols: Genomic DNA Library Protocols

Standard techniques are used here to isolate cytoplasmic RNA from COS cells transfected with a library of recombinant plasmids containing the genomic DNA of interest. - [Read Harvesting the mRNA for Exon Trapping and Amplification]

Category: Immunology: Immunoprecipitation Protocols: Immunoprecipitation of Radiolabeled Cells Protocols

Coimmunoprecipitation is most commonly used to test whether two proteins of interest are associated in vivo, but it can also be used to identify novel interacting partners of a target protein. In both cases, the cells, which may have been labeled with [35S]methionine, are harvested and lysed under conditions that preserve protein-protein interactions. The target protein is specifically immunoprecipitated from the cell extracts, and the immunoprecipitates are fractionated by SDS-PAGE. - [Read Identification of Associated Proteins by Coimmunoprecipitation Protocol]

Category: Western Blot Protocols

Protein immunoprecipitation can be a useful preparative step for immunoblotting.  For very rare proteins, the protein of interest can be purified and concentrated by standard immunoprecipitation techniques before immunoblotting.  In addition, protein-protein interactions can be tested with an immunoprecipitating antibody that is specific for one protein of a complex and an immunoblotting antibody that is specific for a second member of the complex. - [Read Immunoblotting: Preparing Immunoprecipitated Proteins Protocol]

Category: Immunology: Immunoprecipitation Protocols

Protocol for immunoprecipitation of mRNA-protein complexes. In this protocol, an antibody targeting an RBP of interest is used to immunoprecipitate the RBP and any interacting molecules from a cell lysate. Reverse transcription followed by PCR is then used to identify individual mRNAs isolated with the RBP. This method focuses on examining an association between a specific RBP-mRNA complex, and it is best suited for a small scale screening of known or putative binding partners. - [Read Immunoprecipitation of mRNA-Protein Complexes Protocol]

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

LCM utilizes an infrared laser integrated into a standard microscope. A transparent cap is attached to a thermoplastic transparent membrane which lies directly on the surface of a routinely prepared tissue section on a glass slide. The investigator examines the tissue section microscopically and activates the laser when the desired cells underlie the target. This in turn activates the membrane with subsequent binding and procurement of the cells of interest. - [Read Laser Capture Microdissection (LCM)]

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

LCM technology can harvest the cells of interest directly or can isolate specific cells by cutting away unwanted cells to give histologically pure enriched cell populations. A variety of downstream applications exist: DNA genotyping and loss-of-heterozygosity (LOH) analysis, etc. Protocol provides a thorough description of LCM techniques, with an emphasis on tips and troubleshooting advice derived from LCM users. The total time required to carry out this protocol is typically 1–1.5 h. - [Read Laser-capture Microdissection Protocol]

Category: Nucleic Acid Purification Protocols

Preparations of RNA containing an mRNA of interest are hybridized to a complementary single-stranded DNA probe.  At the end of the reaction nuclease S1 is used to degrade unhybridized regions of the probe, and the surviving DNA-RNA hybrids are then separated by gel electrophoresis and visualized by autoradiography or Southern hybridization.  Method used to quantitate RNAs, to map the positions of introns, and to identify the locations of 5' and 3' ends of mRNAs on cloned DNA templates. - [Read Mapping RNA with Nuclease S1 Protocol]

Category: Nucleic Acid Modification Protocols: Nucleic Acid Radiolabeling Protocols

Preparations of RNA containing an mRNA of interest are hybridized to a radiolabeled single-stranded RNA probe. The method can be used to quantitate RNAs, to map the positions of introns, and to identify the locations of 5' and 3' ends of mRNAs on cloned DNA templates. - [Read Mapping RNA with Ribonuclease and Radiolabeled RNA Probes Protocol]

Category: Plant Biology Protocols

The standard protocol for in situ hybridizations in plants still involves fixing fresh tissue, embedding the tissue in wax, sectioning with a microtome and detection of the transcripts of interest using labeled RNA-probes. This protocol concentrates only on nonradioactive methods, as they are easy to perform, very sensitive and even faster than techniques involving radioisotope labels. - [Read Molecular and Biochemical Analysis of Arabidopsis Protocol]

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]