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Tetrad Dissection Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4181

Category: Yeast Cell Culture Protocols: Yeast Sporulation Protocols

This protocol describes dissection of yeast tetrads. Tetrad analysis is useful for linkage studies and for constructing strains for genetic and biochemical experiments. - [Read Tetrad Dissection Protocol]

The Use of Flow Cytometry for Concomitant Detection of Apoptosis and Cell Cycle Analysis - http://www.roche-applied-science.com/PROD_INF/BIOCHEMI/No.2_96/pp25-27.pdf

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: Apoptosis Cytometry Protocols

Two distinct modes of cell death, apoptosis and necrosis, can be distinguished on the basis of differences in morphological, biochemical, and molecular changes occurring in the dying cells. Report the development of flow cytometric techniques that permit concomitant detection of apoptosis and cellular DNA content or BrdU content analysis. - [Read The Use of Flow Cytometry for Concomitant Detection of Apoptosis and Cell Cycle Analysis]

Timeline for Transgenic Mouse Analysis - http://www.med.umich.edu/tamc/tgtime.html

Category: Transgenic Mouse Protocols

Timeline for Transgenic Mouse Analysis and production. University of Michigan Transgenic Animal Model Core. Transgenic Animal Web. - [Read Timeline for Transgenic Mouse Analysis]

Total RNA isolation (Guanidinium Thiocyanate-Phenol-Chloroform Extraction) Protocol - http://www.flemingtonlab.com/Protocols/TotalRNAprep.pdf

Category: Nucleic Acid Purification Protocols: RNA Isolation Protocols

Protocol for total RNA isolation. This method is fast and works well for preparation of total RNA. We typically use total RNA for quantitative S1 nuclease analysis of endogenous (or in vitro) gene expression, and this method works well for this purpose. - [Read Total RNA isolation (Guanidinium Thiocyanate-Phenol-Chloroform Extraction) Protocol]

Transcript In Situ Hybridization of Whole-Mount Embryos for Phenotype Analysis of RNAi-Treated - http://www.cshprotocols.org/cgi/content/full/2006/21/pdb.prot4519

Category: Cytogenetics Protocols: In Situ Hybridization Protocols

This protocol fixes and prepares embryos for in situ hybridization to visualize transcript expression patterns. It is a modification of the method developed by Tautz and Pfeifle for whole-mount in situ analysis of embryos. Use of the standard hybridization protocol on RNAi-treated embryos results in high background staining, which makes visualization of transcript expression patterns practically impossible. The following modifications eliminate this problem and allow visualization of transcript. - [Read Transcript In Situ Hybridization of Whole-Mount Embryos for Phenotype Analysis of RNAi-Treated]

Transcript Profiling by Microarray Analysis Protocol. - http://www.signaling-gateway.org/data/cgi-bin/ProtocolFile.cgi/afcs_PP00000019.pdf?pid=PP00000019

Category: Microarray Protocols: Microarray Hybridization Protocols

Protocol details the preparation of fluorescently labeled target samples and hybridization of these samples to a microarray of Agilent inkjet-deposited cDNAs. The procedure requires a minimum of 5 mg of purified total RNA as starting material. Includes: First Strand cDNA Synthesis; Second Strand cDNA Synthesis; cDNA Cleanup and Precipitation; In vitro Transcription; Cleanup and Quantification of in vitro Transcribed RNA; Fluorescent Labeling of the Target Samples. - [Read Transcript Profiling by Microarray Analysis Protocol.]

Transfection of Bone Marrow-Derived Mast Cells for Transcription Factor Luciferase Reporter Assays - http://www.natureprotocols.com/2006/09/12/transfection_of_bone_marrowder_1.php

Category: Reporter Gene Assays: Luciferase Assay Protocols

Transfection of primary leukocytes has traditionally been a challenging but much desired protocol. It allows not only the analysis of cells in a more natural state to a cell line system, it enables the direct comparison of, for e.g. transcriptional activity using luciferase reporters, in immune cells taken from genetically-altered mice. In addition, importantly it allows for "rescue experiments" in knockout cells & the ability to over-express or reconstitute wild-type and/or mutated constructs. - [Read Transfection of Bone Marrow-Derived Mast Cells for Transcription Factor Luciferase Reporter Assays]

Transgenic Outline: Mice and Rats - http://www.med.umich.edu/tamc/tgoutline.html

Category: Transgenic Mouse Protocols

Clone the Transgene, transgene Screening, Transgene Expression, DNA microinjection, Identify Transgenic Founders, Transgenic Analysis. Transgenic Animal Web. - [Read Transgenic Outline: Mice and Rats]

Two-hybrid analysis of genetic regulatory networks - http://proteome.wayne.edu/YTHnetworks.html

Category: Molecular Model Organisms: Yeast Two-Hybrid Screen Protocol

Finley and Brent. Excellent Two-Hybrid Resource Article and Protocols. Very Detailed. - [Read Two-hybrid analysis of genetic regulatory networks]

Using FLAG Epitope-Tagged Proteins for Coimmunoprecipitation of Interacting Proteins Protocol - http://www.cshprotocols.org/cgi/content/abstract/2006/28/pdb.prot4557

Category: Immunology: Immunoprecipitation Protocols: Protein Immunoprecipitation Protocols

Protocol describes the use of FLAG-epitope-tagged proteins for both small-scale analysis and large-scale coimmunoprecipitation of interacting proteins. When examining protein interactions, it is sometimes possible to immunoprecipitate an endogenous protein X directly, without using an epitope tag, if antibodies are available. The advantage of examining interactions of endogenously expressed proteins is that these are likely to be physiological and less likely to be an artifact of overexpression. - [Read Using FLAG Epitope-Tagged Proteins for Coimmunoprecipitation of Interacting Proteins Protocol]

UV Spectrophotometric Analysis of DNA and RNA - http://www.people.hofstra.edu/faculty/Beverly_Clendening/Adv_Molecular_Biology/Protocols/UV_Spec_Analysis_RNA&DNA.htm

Category: General Research Protocols and Methods: Spectrophotometry Protocols

Determining the concentration of a RNA or DNA sample by using UV spectrophotometry. Hofstra Univ. Beverly Clendening. - [Read UV Spectrophotometric Analysis of DNA and RNA]

Western Analysis Using the Chemiluminescent Alkaline Phosphatase Substrate CSPD Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/16/pdb.prot4256

Category: Western Blot Protocols: Antigen Detection Methods Protocols

Use of the chemiluminescence-producing alkaline phosphatase substrate 3-(4-methoxyspiro[1,2-dioxetane-3,2'-tricyclo-[3.3.1.1(3,7)]decan]-4-yl)phenyl phosphate (AMPPD, also known as adamantyl-1,2-dioxetane phosphate), or its dioxetane relatives provides a substantial increase in sensitivity over colorimetric substrates and radiochemical methods currently used for the detection of antigen-antibody complexes immobilized on nylon or PVDF membranes. - [Read Western Analysis Using the Chemiluminescent Alkaline Phosphatase Substrate CSPD Protocol]

Western Blot Analysis of Endogenous Gene Expression Protocol - http://www.flemingtonlab.com/Protocols/WestBlotAnalyofEndogProts.pdf

Category: Western Blot Protocols

Procedure describes how it denatures most of the modification and degradation proteins immediately giving the most accurate read out of the true levels of protein at the time of harvest. However, in cases where detection is a problem, a limited purification (e.g. isolation of nuclear extract for the detection of transcription factors) might be required to allow analysis. - [Read Western Blot Analysis of Endogenous Gene Expression Protocol]

Western Blot Analysis of Sub-Cellular Fractionated Samples Using the Odyssey Infrared Imaging System - http://www.natureprotocols.com/2006/09/15/western_blot_analysis_of_subce.php

Category: Cell Organelle Protocols: Subcellular Fractionation Protocols

This protocol describes nuclear and cytoplasmic fractionation of tissue culture cells and a method for Western blot detection of proteins using the Odyssey Infrared Imaging System. This protocol was used to detect expression of the "small" Tap protein in 293T, HeLa and COS cells. The Odyssey system has several advantages over the more widely used chemiluminescent detection methods. - [Read Western Blot Analysis of Sub-Cellular Fractionated Samples Using the Odyssey Infrared Imaging System]

Whole-Mount Preparations for Analysis of Axonal Pathfinding in dsRNA-Treated Avian Embryos - http://www.cshprotocols.org/cgi/content/abstract/2006/23/pdb.prot4506

Category: RNAi Technology Protocols: RNAi Avian Embryos Protocols

Protocol describes a method for staining nerve fibers in whole-mount preparations of avian embryos using an antibody against the 160-kD subunit of neurofilaments. This allows the comparison of the branching pattern of motor and sensory neurons between control and experimental embryos. This protocol has been successfully applied for embryos at different stages up to about stage 33 (7 days of incubation). - [Read Whole-Mount Preparations for Analysis of Axonal Pathfinding in dsRNA-Treated Avian Embryos]

Whole-Mount Preparations for Analysis of Axonal Pathfinding in dsRNA-Treated Avian Embryos Protocol - http://www.cshprotocols.org/cgi/content/abstract/2006/23/pdb.prot4506

Category: Avian Bird Protocols

Yeast Protein Extracts Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4152

Category: Yeast Protocols: Yeast Protein Protocols

Protocol is a simple, reliable method for the preparation of yeast protein extracts for analysis by polyacrylamide gel electrophoresis (PAGE) and Western blotting. - [Read Yeast Protein Extracts Protocol]

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Populational Analysis of Genomic DNA Protocol

A protocol on the populational Analysis of genomic DNA.

In Vitro Translated Xenopus Mos Kinase Assay Protocol

In Vitro Translated Xenopus Mos Kinase Assay Protocol. In response to progesterone, immature Xenopus oocytes mature to eggs that can be fertilized. The Mos protein kinase is essential for oocyte maturation, most likely due to its ability to activate the MAP kinase cascade. This MAP kinase cascade eventually leads to the activation of Cdc2/cyclin B and entry into M phase. In this protocol, tagged Mos kinase is translated in vitro, immunopurified, and used in a kinase assay.

Absorption Spectroscopy and Quantification of Filamentous Phage Protocol

Unlike spherical phage, such as T4 and λ, which have roughly equal weight ratios of protein to DNA, filamentous phage have about six times more protein than DNA; the protein therefore contributes substantially to the absorption spectrum.

3' Rapid Amplification of cDNA Ends RACE Using PCR Protocol

3' Rapid Amplification of cDNA Ends RACE Using PCR Protocol. This protocol contains the steps for 3' end rapid amplification of mRNA by PCR. The first-strand cDNA is synthesized from total or poly(A+) RNA by priming from the poly-A tail of the mRNA using a oligo (dT) adaptor primer. The cDNA is then amplified via PCR using a gene-specific primer and an adaptor primer.

Toluidine Blue Stain

Toluidine Blue staining method and protocol for cell or tissue histology.

Thionin Stain

Thionin staining method and protocol for cell or tissue histology.

Picking Transfected ES Cell Clones Protocol

This protocol a protocol on how to generate transfected embryonic stem (ES) cell clones. The previous protocol in this series is the Protocol for Electroporation of ES cells. The next protocol in the series is the Protocol on Disaggregation, Expansion, and Freezing of Transfected ES Clones.

Vector Design Protocol for Transgenic Mice Generation

The protocol gives general considerations for the design of targeting vectors for transgenic mice. The protocol shares tips in the design of knock-out and knock-in vectors and some of their strategies for producing homologously recombined embryonic stem cells.

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