resulting Protocols

Category: Molecular Biology Protocols: Carbohydrate Protocols

The ABH crosslinker binds to the Fc portion of an antibody molecule, away from the antigen binding site, resulting in a divalent immunologically active immunoglobulin derivative. Pierce - [Read ABH (p-Azidobenzoyl hydrazide) PDF]

Category: Cell Culture Protocols: Insect Cell Culture Protocols

This protocol is designed to adapt High Five™ (BTI-Tn-4) cells to serum-free suspension culture in HyQ SFX-Insect in 5 passages. The entire adaptation process takes approximately two weeks and the resulting adapted cells have doubling times of 16 and 20 hours and minimal clumping in suspension culture. - [Read Adaptation of High Five™ Cells to HyQ© SFX Insect Medium]

Category: Cell Biology and Cell Culture: Apoptosis Protocols: Apoptosis Analysis

Analysis of DNA Fragmentation Using the JAM Assay. By Shailaja Kasibhatla et al., The JAM assay is based on labeling nuclear DNA of cycling cells with [3H]thymidine and harvesting samples on glass fiber filters. Apoptosis will generate DNA fragments small enough to pass through the glass fiber filter, resulting in decreased radioactivity of the particular sample. Cell-mediated cytotoxicity or cell killing mediated by cytotoxic T lymphocytes (CTL) can also be measured by this technique. - [Read Analysis Of DNA Fragmentation Using The JAM Assay (Subscription Required)]

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]

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]

Category: Mouse Protocols: Assembling Chimeras Mouse Protocols

Protocol describes a method for producing diploid embryo-tetraploid embryo chimeras. It requires the timed combination of four-cell-stage tetraploid embryo production and the procedure for diploid embryo-diploid embryo aggregation. The resulting chimeras are useful for phenotypic analysis when an induced mutation has an extraembryonic phenotype. - [Read Assembling Aggregates between Diploid and Tetraploid Embryos Protocol]

Category: Mouse Protocols: Assembling Chimeras Mouse Protocols

Protocol describes a method for assembling aggregates between diploid embryos. If embryos from a heterozygous mutant intercross are aggregated with wild-type embryos, the resulting chimeras can be used for analyzing mutant phenotypes. - [Read Assembling Aggregates between Diploid Embryos Protocol]

Category: Mouse Protocols: Assembling Chimeras Mouse Protocols

Protocol describes a method for assembling aggregates between ES cells and diploid embryos. The resulting chimeras are useful for separating certain extraembryonic phenotypes from phenotypes in the embryo proper, since the diploid embryo contributes to all parts of the conceptus, but the ES cell component does not contribute to the trophoblast or yolk sac endoderm. - [Read Assembling Aggregates between Embryonic Stem (ES) Cells and Diploid Embryos Protocol]

Category: Mouse Protocols: Assembling Chimeras Mouse Protocols

Protocol describes a method for producing ES cell-tetraploid embryo chimeras. It requires the timed combination of four-cell-stage tetraploid embryo production and the procedure for ES cell-diploid embryo aggregation in which diploid embryos are replaced with tetraploid embryos. The resulting chimeras can be used to analyze the embryonic versus extraembryonic phenotype of a mutation. - [Read Assembling Aggregates between Embryonic Stem (ES) Cells and Tetraploid Embryos Protocol]

Category: RNA Protocols: RT-PCR and cDNA synthesis

Protocol for Competitive RT-PCR.For quantifying mRNA, we use a competitive RT-PCR protocol with internal standard RNAs. These are added in a defined quantity to the RNA sample prior to the RT reaction. The resulting standard cDNA is coamplified with the s - [Read Competitive RT-PCR Protocol]

Category: Microinjection Protocols: Microinjection of DNA Protocols

For microinjection into cells, high-quality plasmid DNA is purified using standard procedures. The resulting preparation is then delivered into the microinjection needle by either a back-filling or a forward-filling approach. - [Read DNA Preparation and Needle Loading for Gene Delivery by Microinjection Protocol]

Category: E Coli Protocols: E coli Genetics Protocols

Protocol for the generation of gene deletions and gene replacements in Escherichia coli O157:H7 using a temperature sensitive allelic exchange system. Technology requires flanking DNA to be cloned into a temperature sensitive vector but the resulting clone allows great flexibility for further modification of the target sequence. It is therefore highly suited to the study of genes in which several rounds of changes are envisaged. - [Read Generation of Gene Deletions and Gene Replacements in Escherichia coli Protocol]

Category: Immunology: Immunoprecipitation Protocols

For many sources of antigens, one useful method of lysis is to treat cells with harsh, denaturing solutions to release most of the protein antigens, as described here. The lysates are then diluted to reduce the denaturing conditions to levels that are suitable for the formation of antibody-antigen complexes. The resulting solution is precleared prior to immunoprecipitation. - [Read Immunoprecipitation: Denaturing Lysis Protocol]

Category: Bacterial Cell Culture Protocols

In this protocol, bacterial cells are lysed by being subjected to short, intense treatments with ultrasound, which breaks the cell walls and shears the DNA into sizes that will not affect the viscosity of the samples. Note that this method causes some denaturation of the samples. The resulting lysate is ready for preclearing. - [Read Immunoprecipitation: Lysing Bacteria by Sonication Protocol]

Category: Immunology: Mononuclear Cell Isolation Protocols

Presents two methods for preparing dendritic cells (DCs), a highly specialized type of antigen-presenting cell (APC). The first method involves the isolation of DCs from mouse spleen, resulting in a cell population that is highly enriched in accessory cell and APC function. A support protocol for collagenase digestion of splenocyte suspensions is described to increase the yield of dendritic cells. The second method involves generating large numbers of DCs from mouse bone marrow progenitor cells. - [Read Isolation of Dendritic Cells Protocol]

Category: Yeast Protocols: Yeast Nucleic Acid Protocols: Yeast DNA Isolation Protocols

Protocol describes the purification release of plasmid DNA from yeast cells. The resulting plasmid DNA can be used to transform Escherichia coli or yeast. - [Read Isolation of Plasmid DNA from Yeast Cells: A Ten-Minute Preparation]

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]

Category: Viral Culture and Isolation Protocols: Viral Culture Protocols

In this protocol, a bacterial lysogen is constructed from a recombinant bacteriophage {lambda} encoding a fusion protein of interest. The resulting lysogenic colonies are induced to synthesize the fusion protein, which is then isolated in preparation for functional and biochemical analyses. - [Read Preparation of Lysates Containing Fusion Proteins Encoded by Bacteriophage {lambda} Lysogens]

Category: Plant Biology Protocols: Plant DNA RNA Isolation Protocols

Tissues are homogenized in guanidinium isothiocyanate lysis buffer and then subjected to cesium chloride ultracentrifugation. The resulting RNA is then extracted with phenol and chloroform to remove protein and lipid contaminants. - [Read Preparation of RNA from Plant Tissue Using Guanidinium Isothiocyanate/Cesium Chloride Ultracentrifug]

Category: Cell Biology and Cell Culture

Purified Tubulin is polymerized and then stabilized with Taxol. The resulting Microtubules are then ready for use in motility assays. Protocol use solutions such as 50 mM Mg-GTP, 50 μl 100 mM GTP, 40 μM Taxol, Taxol Stock,and PM Buffer. - [Read Protocol for Microtubule Assembly]

Category: Cell Organelle Protocols: Subcellular Fractionation Protocols

This protocol is employed for the purification of a population of muscle-derived cells from skeletal muscle of C57Bl/6 animals. The resulting preparation is a mixture of many cell types including satellite cells (a.k.a., muscle progenitor cells) and hematopoietically active muscle-derived cells. - [Read Protocol for the Isolation of a Heterogenous Muscle-Derived Cell Population]

Category: Nucleic Acid Electrophoresis Protocols: DNA Electrophoresis Protocols: DNA Electrophoresis in Agarose Gels Protocols

DNA fragments separated by electrophoresis through gels cast with low-melting-temperature agarose are recovered by melting the agarose and extracting the resulting solution with phenol:chloroform.  The protocol works best for DNA fragments ranging in size from 0.5 kb to 5 kb. - [Read Recovery of DNA from Low-melting-temperature Agarose Gels: Organic Extraction Protocol]

Category: Nucleic Acid Electrophoresis Protocols: Pulsed Field Gel Electrophoresis Protocols

Protocol for retrieval of DNA fragments from pulsed-field gels following DNA concentration. DNA contained in a slice of low-melting-temperature agarose is first concentrated by electrophoresis into a high-percentage agarose gel, and then isolated by treatment with agarase.  The resulting DNA preparation is purified by microdialysis. - [Read Retrieval of DNA Fragments from Pulsed-field Gels following DNA Concentration Protocol]

Category: Nucleic Acid Electrophoresis Protocols: RNA Electrophoresis Protocols

Separation of RNAs according to size is the first stage in northern blotting and hybridization.  The method described in this protocol uses glyoxal to denature the RNA, ethidium bromide to stain it, and agarose gel electrophoresis to separate the resulting glyoxal-RNA-ethidium adducts. - [Read Separation of RNA According to Size: Electrophoresis of Glyoxylated RNA through Agarose Gels]

Category: Nucleic Acid Electrophoresis Protocols: RNA Electrophoresis Protocols

Separation of RNAs according to size is the first stage in northern blotting and hybridization.  The method described in this protocol uses formaldehyde to denature the RNA, ethidium bromide to stain it, and electrophoresis through agarose gels containing 2.2 M formamide to separate the resulting formaldehyde-RNA-ethidium adducts. - [Read Separation of RNA According to Size: Electrophoresis of RNA through Agarose Gels Containing Formalde]