combination Protocols

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 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: Cell Biology and Cell Culture: Flow Cytometry Protocols: Cell Sorting Protocols

The combination of prospective identification/isolation of bone marrow progenitors and quantitative RT-PCR is a powerful tool to understand the molecular mechanism underlying hematopoiesis. Describes the standard procedures of the murine myeloid progenitor staining for fluorescence activated cells sorting (FACS) and RNA purification methods. - [Read Cell Staining for Sorting of Hematopoietic Stem Cells (HSC) and Myeloid Progenitors]

Category: Stem Cell Protocols

The combination of prospective identification/isolation of bone marrow progenitors and quantitative RT-PCR is a powerful tool to understand the molecular mechanism underlying hematopoiesis. Here, we described our standard procedures of the murine myeloid progenitor staining for fluorescence activated cells sorting (FACS) and RNA purification methods. - [Read Cell Staining for Sorting of Hematopoietic Stem Cells and Myeloid Progenitors and Isolating RNA]

Category: Reporter Gene Assays: GFP Assay Protocols

Specific molecular components can be efficiently labeled by a combination of three methods: chemical transfection of GFP-fusion constructs, staining of chromosomes with the DNA-specific, fluorescent dye Hoechst 33342, and microinjection of fluorescently conjugated proteins. This procedure provides an example of using all three methods in sequence to label components of living HeLa cells. These methods should be followed in the order presented, but any of them can be omitted when not needed. - [Read Imaging Hoechst-Labeled Chromosomes and Fluorescent Proteins during the Cell Cycle]

Category: Microinjection Protocols

Fluorescence microscopy provides a powerful tool for imaging molecular components in living cells. Specific molecular components can be efficiently labeled by a combination of three methods: chemical transfection of GFP-fusion constructs, staining of chromosomes with the DNA-specific, fluorescent dye Hoechst 33342, and microinjection of fluorescently conjugated proteins. This procedure provides an example of using all three methods in sequence to label components of living HeLa cells. - [Read Imaging Hoechst-Labeled Chromosomes and Fluorescent Proteins during the Cell Cycle]

Category: Plant Biology Protocols: Plant Genetics Protocols

The preparation of expressional cDNA libraries for use in the yeast two-hybrid system is quick and efficient when using the dedicated Clontech™ product, the MATCHMAKER Library Construction and Screening Kit 3. This kit employs SMART technology for the amplification of full-length cDNAs, in combination with cloning using homologous recombination. - [Read Isolation of Plant Transcription Factors Using a Modified Yeast One-Hybrid System]

Category: Cytogenetics Protocols

Protocol describes a recently developed method — methylation-specific digital karyotyping (MSDK) — that enables comprehensive and unbiased genome-wide DNA methylation analysis. Using a combination of a methylation-sensitive mapping enzyme (for example, AscI) and a fragmenting enzyme (for example, NlaIII), short sequence tags can be obtained and uniquely mapped to genome location. - [Read Methylation-Specific Digital Karyotyping Protocol]

Category: Chromatography Protocols: Dye Ligand Affinity Chromatography Protocols

Protocol for the optimization of elution condition for dye-ligand affinity chromotography. Elution methods used in dye-ligand affinity chromatography may be either selective or nonselective in nature.  Usually, selective elution methods are applied in combination with group-specific adsorbents, such as dye-ligand adsorbents, and nonselective elution methods are used in combination with highly specific adsorbents. - [Read Optimization of Elution Conditions for Dye-Ligand Affinity Chromatography Protocol]

Category: Mouse Protocols: Mouse Reproductive System Surgery Protocols

Protocol describes a method for removal of mouse ovaries in combination with the administration of progesterone, which causes the arrest of blastocyst development. This procedure is performed during the afternoon of the third day of pregnancy to ensure that the morulae have moved sufficiently into the oviducts and will be less likely to be damaged during the ovariectomy. - [Read Ovariectomy for Induction of Blastocyst Implantation Delay Protocol]

Category: Chromatography Protocols: Affinity Chromatography Protocols

Protein complexes can be isolated by several different approaches.  For example, a protein can be tagged with an epitope such as Flag or TAP and then overexpressed in a target cell, allowing the interacting proteins to be purified.  Similarly, epitope tags can be homologously recombined into the endogenous locus ("knocked-in"), allowing protein complexes containing the tagged proteins to be isolated at their natural expression level. - [Read Overview of Affinity Purification in Combination with Mass Spectrometry Protocol]

Category: Drosophila Protocols: Drosophila Staining Protocols

Early embryos (0-17 hours or until cuticle formation) are treated with a mixture of organic solvents, formaldehyde, and alcohols, as described here. The cuticles of late-stage embryos are usually opened by sonication. Tissues from more advanced stages of development are normally dissected by hand and then fixed and stained in a standard paraformaldehyde/detergent combination - [Read Preparing Early Whole-Mount Drosophila Embryos for Immunostaining Protocol]

Category: Drosophila Protocols: Drosophila Staining Protocols

Early and late embryos are treated with a mixture of organic solvents, formaldehyde, and alcohols. The cuticles of late-stage embryos (17-22 hours or until hatching) are usually opened by sonication, as described here. Tissues from later stages of development are normally dissected by hand and then fixed and stained in a standard paraformaldehyde/detergent combination. - [Read Preparing Late Whole-Mount Drosophila Embryos for Immunostaining Protocol]

Category: Protein Protocols: Protein Extraction From Tissue

Combination of nucleic acid and protein isolation with tissue array construction: Using defined histologic regions in single frozen tissue blocks for multiple research purposes - [Read Protein isolation with tissue array construction]

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

Protocol for pulsed-field gel electrophoresis via contour-clamped homogeneous electric field gels. In CHEF gels, the electric field is generated from multiple electrodes, arranged in a square of hexagonal contour around the horizontal gel and clamped to predetermined potentials.  Using a combination of low field strengths, low concentrations of aragose, long switching intervals, and extended periods of electrophoresis, DNAs up to 5000 kb can be resolved. - [Read Pulsed-field Gel Electrophoresis via Contour-clamped Homogeneous Electric Field Gels Protocol]

Category: Chromatography Protocols: DNA RNA Affinity Chromatography

When many RNA samples are to be processed or when working with small amounts (<50 µg) of total mammalian RNA, the technique of choice is batch chromatography on oligo(dT)-cellulose. The method described in this protocol uses a combination of temperature and ionic strength to maximize binding and recovery of polyadenylated RNA. IMPORTANT: Prepare all reagents used in this protocol with Diethyl pyrocarbonate (DEPC)-treated H2O. Joseph Sambrook and David W. Russell. - [Read Selection of Poly(A)+ RNA by Batch Chromatography - Subscription Required]

Category: Xenopus Protocols: Xenopus Genetics Protocols

Protocols for performing wholemount fluorescent in situs. Instructions for two-color FISH and for a combination of fluorescent and colorimetric in situs we have called FCIS. Data can be imaged with a fluorescence stereoscope or a confocal. - [Read Xenopus Fluorescent in situs and FCIS Protocol]