performed Protocols

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: Genetics and Genomics: Cancer Genetics Protocols: Loss of Heterozygosity Protocols

DNA for analysis is purified using salt precipitation. The method is gentle, limits the breakage of the long chromosomal strands, and avoids the use of phenol and chloroform. It is suitable for use with cultured cells, breast tumor tissue that has been subjected to hormone receptor analysis, and blood samples. The loss of heterozygosity assay is performed using a multiplex PCR, in which one of each primer pair is labeled with a different fluorophor. - [Read A Multiplex PCR Method to Define a Narrow Deleted Chromosomal Region of a Tumor Genome]

Category: Epigenetics and Methylation Protocols: ChIP Chromatin Immunoprecipitation Protocols

Chromatin Immunoprecipitation Protocol for Histone Modification Chromatin and Associated Proteins. Roderick O’Sullivan & Joost Martens. Chromatin Immunoprecipitation (ChIP) experiments are routinely performed in many laboratories around the world to examine the occupancy of proteins or chromatin modifications over particular stretches of the genome. - [Read Chromatin Immunoprecipitation Protocol for Histone Modification Chromatin and Associated Proteins]

Category: Genetics and Genomics: Cancer Genetics Protocols

Investigators can utilize X chromosome inactivation (methylation) to determine the clonality status of a tumor or premalignant lesion in females. The technique is based on a methylation-sensitive restriction enzyme and analysis of a polymorphic locus on the X chromosome. Clonal cell populations will show "loss" of the non-methylated allele after restriction digest. The assay can be performed on DNA recovered from microdissected samples. Both frozen tissue and fixed-embedded tissue can be used. - [Read Clonality - X Chromosome Inactivation Assay Protocol]

Category: Oligonucleotide Protocols: Oligonucleotide Purification Protocols

Concentration of DNA by Ethanol Precipitation Protocol. Adapted from Bruce A. Roe, Department of Chemistry and Biochemistry, The University of Oklahoma, Norman, Oklahoma. Usually 2.5 - 3 volumes of  ethanol and/or acetate solution is added to the DNA in a microcentrifuge tube. This is then put into an ice-water bath for at least 10 minutes. The precipitation is performed by incubation at -20C overnight. - [Read Concentration of Oligo DNA by Ethanol Precipitation Protocol]

Category: Microscopy Protocols: Fluorescence Microscopy Protocols

Certain fluorescent dyes such as Blankophor have a high affinity for the b -glycosidically linked polysaccharides such as glucan and chitin, which are main the constituents of the fungal cell wall. Therefore, these fluorescent dyes can be used for screening clinical samples for the presence of fungal elements. This procedure can be performed using the following specimens: Nail, Skin, Bronchial alveolar lavage fluid, Sputum and Biopsies. - [Read Detection of Fungi by Fluorescence Microscopy Using Fluorescent Brighteners]

Category: Yeast Protocols: Yeast Protein Protocols

This assay is performed to detect ubiquitylated proteins in yeast. Yeast that have been transformed with a vector expressing polyhistidine-tagged ubiquitin (Ub) under the control of a copper-inducible promoter are grown, induced with copper, and harvested. Total ubiquitylated proteins are then recovered by nickel-affinity chromatography, and specific proteins are detected by Western blotting. - [Read Detection of Ubiquitylated Proteins in Yeast Protocol]

Category: Molecular Biology Protocols: DNA Ligation Protocols

DNA ligations are performed by incubating DNA insert with linearized cloning vector in the presence of ligation buffer, rATP, and T4 DNA ligase. Roe. Univ. Oklahoma. - [Read DNA ligation with Linearized DNA]

Category: Mouse Protocols: Mouse Genetics Protocols

Protocol describes a method for electroporating DNA into ES cells, as well as selection methods. Pilot studies should be performed to optimize the conditions for each DNA construct. The selection method described here is one of the most complex. It involves targeting constructs in which the bacterial neomycin-resistance gene disrupts the coding sequence of the mouse gene. - [Read Electroporating DNA into Embryonic Stem (ES) Cells and Selection Methods Protocol]

Category: Immunology: Macrophage Monocyte Protocols

Erythrophagocytosis Assay. The erythrophagocytosis assay is performed to compare the phagocytic rate with and without anti-eythrocyte antibody in either macrophages from control animals or virus-infected counterparts. Andrei Musaji Viral Immunity and Pathogenesis Group - [Read Erythrophagocytosis Assay]

Category: Viral Culture and Isolation Protocols: Viral Culture Protocols

A crude lysate gel assay can be performed to roughly quantitate the DNA in lysates. This is often a valuable time saving step to determine if the phage yield is sufficient to warrant continuing the procedure. - [Read Gel Assay to Determine DNA Content of Phage Lysates Protocol]

Category: Plant Biology Protocols: Arabidopsis Genetics Protocols

Protocol describes the use of glufosinate ammonium to select transformed Arabidopsis plants. The major advantage of glufosinate ammonium selection is that it can be performed on plants growing in soil and does not require the use of sterile techniques. - [Read Glufosinate Ammonium Selection of Transformed Arabidopsis Protocol]

Category: Immunology: T Cell Protocols

Protocol for the Stimulation of human peripheral blood mononuclear cells with anti-human CD3 monoclonal antibody; MTT assay for detection of cellular proliferation. Human PBMCs can be activated in vitro by soluble anti-human CD3 antibodies. Performed titration studies with these antibodies and established the following protocol for stimulation of PBMC. - [Read Human T Cell Activation Protocol]

Category: Primary Cell Isolation and Culture Protocols: Mammalian Cell Culture Protocols

This procedure describes the isolation and culture of adult mouse cardiac myocytes from two or more hearts. Includes modifications for the digestion of two or more hearts in the same procedure and subsequent pooling of myocytes derived from the multiple hearts. The isolation procedure is performed by one or more technicians and routinely yields approximately 1 million rod-shaped myocytes per heart. - [Read Isolation of Adult Mouse Cardiac Myocytes from Two or More Hearts Protocol]

Category: Yeast Protocols: Yeast Staining Protocols

Protocol describes our method for preparing cells for immunofluorescence, in which all incubations and washes are performed in microtiter dishes. The protocol can also easily be adapted for preparing cells for immunofluorescence in microfuge tubes. - [Read Large-Scale Immunocytology Protocol]

Category: Mouse Protocols: Mouse Reproductive System Surgery Protocols

Protocol describes castration of male mice, which is used to eliminate testicular hormones and/or to obtain testes for analysis without sacrificing the male. Castration is performed in a similar manner to vasectomy. - [Read Mouse Castration Protocol]

Category: Nucleic Acid Detection Protocols: Nucleic Acid Blotting Protocols

Protocol for northern hybridization. Protocol describes how to carry out northern hybridization at high stringency in phosphate-SDS-buffers.  Although a wide variety of formats are available, hybridization is usually performed in heat-sealable bags, roller bottles, or plastic boxes, as described here. - [Read Northern Hybridization 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: Signalling Signal Transduction Protocols

Detection of phosphorylated tyrosine residues can be performed using anti-P-TYR Ab and Western Analysis.Includes 2nd method,which uses phosphotyrosine in conjunction with anti-P-TYR Ab to "unlabel" potential proteins.By comparing Westerns developed with the 1st method(reveals phosphorylated protein) and the 2nd method(reveals non-specific labeling), a more accurate picture of those proteins phosphorylated on tyrosine can be seen. Includes: Protein Preparation, Electrophoresis and Transfer. - [Read Protocol for Antiphosphotyrosine Western Blot Analysis]

Category: Primary Cell Isolation and Culture Protocols: Mammalian Cell Culture Protocols

Cryopreserved PBMCs are a common specimen source for studies of immunological responses to vaccines, immunotherapies, etc. The health and viability of cells recovered post-cryopreservation are of course critical to the success and accuracy of immunological assays performed on them. This protocol standardizes PBMC isolation and cryopreservation techniques, specifically for the assessment of thawed cells by cytokine flow cytometry. - [Read Protocol for Isolation, Cryopreservation, and Thawing of PBMCs]

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

Recovery of bands of DNA from agarose gels by electrophoresis onto a sliver of DEAE-cellulose membrane can be performed simultaneously on many samples and reliably gives high yields of fragments between 500 bp and 5 kb in length. - [Read Recovery of DNA from Agarose Gels: Electrophoresis onto DEAE-cellulose Membranes Protocol]

Category: Cytogenetics Protocols: In Situ Hybridization Protocols

This protocol describes a method for reverse transcriptase (RT) in situ PCR. In situ PCR differs from PCR in situ hybridization in the inclusion of a reporter molecule in the amplification step. The two steps of RT in situ PCR that differ from in situ PCR are overnight digestion in RNase-free DNase that is performed after protease digestion, and an RT step, prior to in situ PCR. - [Read Reverse Transcriptase In Situ PCR Protocol]

Category: PCR Protocols: Reverse Transcriptase RT-PCR Protocols

Protocol describes a method for reverse transcriptase (RT) in situ PCR.  In situ PCR differs from PCR in situ hybridization in the inclusion of a reporter molecule in the amplification step.  The two steps of RT in situ PCR that differ from in situ PCR are overnight digestion in Rnase-free Dnase that is performed after protease digestion, and an RT step, prior to in situ PCR. - [Read Reverse Transcriptase In Situ PCR Protocol]

Category: RNAi Technology Protocols: RNAi C. elegans Protocols

Protocol for RNAi screens in C. elegans in a 96-well liquid format and their application to the systematic identification of genetic interactions. The procedure allows thousands of RNAi feeding experiments to be performed per investigator per day. - [Read RNAi Screens in C. elegans Protocol]

Category: PCR Protocols: Colony PCR

The following colony PCR protocol has been designed to be performed in individual reaction tubes. We usually test three colonies from each transformation along with a wild-type control. A series of five different PCR tests are performed on each colony to - [Read Single tube confirmation PCR protocol]