content Protocols

Category: DNA Protocols: cDNA Library Protocols

Here, the DNA-RNA hybrids synthesized in Stage 1 are converted into full-length double-stranded cDNAs. The primers for synthesis of second-strand cDNA are created by RNase H, which introduces nicks into the RNA moiety of the cDNA-mRNA hybrids. E. coli DNA polymerase I extends the newly created 3'-hydroxyl termini, using the first-strand cDNA as a template. - [Read Construction of cDNA Libraries Protocol]

Category: DNA Protocols: cDNA Library Protocols

The goal of this stage is to introduce methyl groups that will modify and protect naturally occurring EcoRI sites in the double-stranded cDNA. - [Read Construction of cDNA Libraries Protocol.]

Category: DNA Protocols: Cosmid Library Protocols

Protocol describes how to construct a library of 35-45-kb fragments of genomic DNA in the double cos site cosmid vector, SuperCos-1. The steps include: Linearization and dephosphorylation of SuperCos-1 DNA; Partial digestion of high-molecular-weight DNA with MboI; Dephosphorylation of high-molecular-weight genomic DNA; Ligation of cosmid arms to genomic DNA: Packaging and plating recombinants; Isolation and analysis of recombinant cosmids: Validation of the library. - [Read Construction of Genomic DNA Libraries in Cosmid Vectors Protocol]

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]

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]

Category: Reporter Gene Assays: Luciferase Assay Protocols

It is possible that some cell lines have lost the ability to perform RNAi or that cells derived from certain tissues do not support RNAi. This reporter assay, for RNAi in mammalian cells, can be used to establish whether the cells under study are susceptible to RNAi. - [Read Cotransfection of Luciferase Reporter Plasmids with siRNA Duplexes Protocol]

Category: RNAi Technology Protocols: RNAi Mammalian Cells Protocols

This reporter assay, for RNAi in mammalian cells, can be used to establish whether the cells under study are susceptible to RNAi. - [Read Cotransfection of Luciferase Reporter Plasmids with siRNA Duplexes Protocol]

Category: Cell Culture Protocols: Cell Staining Protocols

Counterstains are used to help differentiate the various cell types of subcellular structures seen in cell staining. They are essential for tissue sections, allowing the identification of the cell types, but also may be helpful in other staining reactions. - [Read Counterstains Protocol]

Category: Cell Culture Protocols: Cell Storing and Cell Freezing Protocols

Protocol provides methods for cryofreezing and subsequent thawing of mammalian cells. Pre-confluent cells are trypsinized, pelleted, resuspended in freezing medium, and gradually frozen. When needed, frozen cells are thawed quickly under running tap water and transferred to growth medium. - [Read Cryopreservation of Mammalian Culture Cells: Preparation and Recovery of Samples Protocol]

Category: Plant Biology Protocols

In most natural habitats, Arabidopsis is a winter annual: Its seeds germinate in the fall, the young plants survive the winter, floral meristems emerge in the spring, and only the seeds survive the summer months. Most common laboratory varieties of Arabidopsis flower within 4 weeks of germination, and seeds can be collected after an additional 4-6 weeks. - [Read Cultivation of Arabidopsis Protocol]

Category: Laboratory Model Organisms Protocols

Protocol describes the culture of marine euplotids using Dunaliella salina or D. tetiolecta as a food organism. Dunaliella tolerate a wide range of salinity, thus they are fairly easy to grow in the lab using artificial sea salts. - [Read Culturing Marine Euplotids Using Dunaliella as a Food Source Protocol]

Category: Stem Cell Protocols: Stem Cell Culture Protocols

Culture conditions have been established for a second blastocyst-derived cell line, trophoblast stem (TS) cells, in addition to embryonic stem (ES) cells. This protocol describes a method for culturing TS cell lines. These cells can then be used to study trophoblast differentiation and placental function. - [Read Culturing Trophoblast Stem (TS) Cell Lines Protocol]

Category: Cell Biology and Cell Culture: Apoptosis Protocols

Cytochemical Methods for the Detection of Apoptosis. Mark C. Willingham 1999 - [Read Cytochemical Methods for the Detection of Apoptosis]

Category: Stem Cell Protocols: Stem Cell Isolation Protocols

The starting material for de novo isolation of stem cell lines can be either normal 3.5-days post coitum (dpc) expanded blastocysts or "delayed" blastocysts. Delayed blastocysts are usually collected 4-6 days after ovariectomy. For both groups of blastocysts, tissue culture procedures are similar. The only difference is the timing of the first disaggregation, because delayed blastocysts will initially grow more slowly. - [Read De Novo Isolation of Embryonic Stem (ES) Cell Lines from Blastocysts Protocol]

Category: PCR Protocols

Protocol for dealing with carryover contamination in PCR- enzymatic strategy. Repeated use of PCR and manipulation of its products cause aerosols that can contaminate neighboring samples and work areas.  Such "carryover contamination" can be prevented by including dUTP in place of dTTP for all amplification reactions. - [Read Dealing with Carryover Contamination in PCR: An Enzymatic Strategy Protocol]

Category: RNAi Technology Protocols: RNAi Drosophila Protocols

Protocol describes how subcellular-sized particles are accelerated to high velocity to carry double-stranded RNA (dsRNA) into Drosophila embryos.  The major advantage of this procedure over microinjection (Microinjection of dsRNA into Drosophila Embryos) is that particle bombardment is easier and faster to perform.  In addition, the mechanical trauma received is far less than by microinjection, allowing better survival of embryos and fewer phenotypic artifacts. - [Read Delivery of dsRNA into Drosophila Embryos by Gene Gun 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: Immunology: Immunoprecipitation Protocols: Protein Immunoprecipitation Protocols

Protocol describes a denaturing immunoprecipitation (IP) for mammalian cells. Prefer to use denaturing IPs to recover labeled proteins from pulse-chase experiments. The use of denaturing IPs reduces background considerably. - [Read Denaturing Protein Immunoprecipitation from Mammalian Cells Protocol]

Category: Yeast Protocols: Yeast Protein Protocols: Yeast Immunoprecipitation Protocols

The use of denaturing Ips reduces background and simplifies protein harvest. Yeast are boiled in an SDS-containing solution to liberate total proteins. - [Read Denaturing Protein Immunoprecipitation from Yeast Protocol]

Category: Stem Cell Protocols

This protocol decribes derivation of TS cell lines from 3.5-days post coitum (dpc) mouse blastocysts. The procedure is similar to the derivation of embryonic stem (ES) cell lines. However, the success rate is considerably higher, and less expertise is required to recognize pluripotent TS cell colonies. - [Read Derivation of Trophoblast Stem (TS) Cell Lines from Blastocysts Protocol]

Category: Chromatography Protocols: Liquid Chromatography Protocols

Protocol for desalting of peptides and protein mixtures by RP-HPLC techniques. The RP-HPLC technique can be used to "desalt" peptide or protein samples derived from extraction procedures, from chemical reactions such as reductive alkylation in the presence of urea or guanidine hydrochloride, citraconylation, iodination, or cyanogen bromide cleavage, or recovered from other chromatographic separation. - [Read Desalting of Peptides and Protein Mixtures by RP-HPLC Techniques Protocol]

Category: Microscopy Protocols: Live-Cell Imaging Protocols

When choosing a particular molecule for photoactivation studies, it is necessary to have some structural knowledge of the molecule in order to design an appropriately caged species that will retain its biological inactivity until uncaging is effected. Includes synthesis of caged peptides or proteins. - [Read Design, Synthesis, and Characterization of Caged Compounds]

Category: Microinjection Protocols: Microinjection of Proteins Protocols

When choosing a particular molecule for photoactivation studies, it is necessary to have some structural knowledge of the molecule in order to design an appropriately caged species that will retain its biological inactivity until uncaging is effected. - [Read Design, Synthesis, and Characterization of Caged Compounds Protocol]

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

The most convenient and commonly used method to visualize DNA in agarose gels is staining with the fluorescent dye ethidium bromide.  Ethidium bromide can be used to detect both singleand double-stranded nucleic acids (both DNA and RNA).  However, the affinity of the dye for single-stranded nucleic acid is relatively low and the fluorescent yield is comparatively poor. - [Read Detection of DNA in Agarose Gels Protocol]

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

Detection of radioactive DNA in polyacrylamide gels by autoradiography. - [Read Detection of DNA in Polyacrylamide Gels by Autoradiography Protocol]