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A Single-step Method for the Simultaneous Preparation of DNA, RNA, and Protein from Cells and Tissue - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot4056

Category: Nucleic Acid Purification Protocols

Protocol for a single-step method for the simultaneous preparation of DNA, RNA, and protein from cells and tissues. The yield of total RNA depends on the tissue or cell source, but it is generally in the range of 4-7 µg/mg starting tissue or 5-10 µg/106 cells. IMPORTANT: Prepare all reagents used in this protocol with Diethyl pyrocarbonate (DEPC)-treated H2O. - [Read A Single-step Method for the Simultaneous Preparation of DNA, RNA, and Protein from Cells and Tissue]

Absorbance Assay 280 nm - http://www.ruf.rice.edu/~bioslabs/methods/protein/abs280.html

Category: Protein Protocols: Protein Quantitation Concentration Protocols

Absorbance assays are fast and convenient, since no additional reagents or incubations are required. No protein standard need be prepared. The assay does not consume the protein. The relationship of absorbance to protein concentration is linear. Because different proteins and nucleic acids have widely varying absorption characteristics there may be considerable error, especially for unknowns or protein mixtures. - [Read Absorbance Assay 280 nm]

Accumulation and Fixation of Plant Metaphase Chromosomes Protocol - http://www.le.ac.uk/bl/phh4/proaccum.htm

Category: Plant Biology Protocols: Plant Genetics Protocols

For analysis of metaphase chromosomes, any tissue containing dividing cells can be used: Root tips from young seedlings, from newly grown roots at the edge of plant pots or hydroponic culture are all suitable. Alternatively, flower buds, anthers, carpels or leaf or apical meristems can be used. Includes metaphase arresting reagents. - [Read Accumulation and Fixation of Plant Metaphase Chromosomes Protocol]

Antibody Addition to Drosophila Specimens and Detection Using Enzyme-Linked Reagents Protocol - http://www.cshprotocols.org/cgi/content/abstract/2006/23/pdb.prot4526

Category: Drosophila Protocols

Enzyme-linked reagents give excellent sensitivity and use a simple light microscope for detection. A range of enzymes is available, but for staining in situ, horseradish peroxidase will suit most needs. Diaminobenzidine (DAB) is one of the most sensitive substrates for horseradish peroxidase. It yields an intense brown product that is insoluble in both water and alcohol. It can be made more sensitive by adding metal salts such as cobalt or nickel to the substrate solution. - [Read Antibody Addition to Drosophila Specimens and Detection Using Enzyme-Linked Reagents Protocol]

Antibody Addition to Drosophila Specimens and Detection Using Fluorochrome-Linked Reagents Protocol - http://www.cshprotocols.org/cgi/content/abstract/2006/23/pdb.prot4527

Category: Drosophila Protocols

Protocol for antibody addition to Drosophila specimens and detection using fluorochrome-linked reagents. Fluorochrome-linked reagents should be used when high resolution is needed or if two antigens need to be localized simultaneously. Because of the thickness of fly specimens, detection requires access to a confocal microscope. - [Read Antibody Addition to Drosophila Specimens and Detection Using Fluorochrome-Linked Reagents Protocol]

Antigen Retrieval for Immunohistochemistry with Paraffin-Embedded Tissues Protocol - http://www.animal.ufl.edu/hansen/Protocols/Immunohistochemistry_paraffin.htm

Category: Immunohistochemistry Protocols: Antigen Retrieval Protocols

Protocol used to for immunohistochemistry on paraffin-embedded sections. Based on use of microwave energy to effect antigen retrieval. The immunohistochemistry procedure, is for use of Biomeda's HistoScan kit based on a streptavidin-peroxidase/biotinylated second antibody detection system with 3-amino, 9-ethylcarbazole (AEC) as chromogen. Undoubtedly, other kits or home-made reagents will also work . - [Read Antigen Retrieval for Immunohistochemistry with Paraffin-Embedded Tissues Protocol]

Assay of Cyclic AMP in Lysates of Cells - http://www.signaling-gateway.org/data/cgi-bin/ProtocolFile.cgi/afcs_PP00000015.pdf?pid=PP00000015

Category: Signalling Signal Transduction Protocols

Protocol allows you to measure the content of cyclic adenosine 3',5'-monophosphate (cyclic AMP or cAMP) in splenic B lymphocytes (B cells) in an enzyme-linked immunoassay. This protocol utilizes acetylation of cAMP to improve sensitivity and reduce interference. Protocol includes information on: how to determine cAMP, calculations and reagents and materials. - [Read Assay of Cyclic AMP in Lysates of Cells]

Assay of Cytokines in Tissue Culture Supernatants - http://www.signaling-gateway.org/data/cgi-bin/ProtocolFile.cgi/afcs_PP00000223.pdf?pid=PP00000223

Category: Signalling Signal Transduction Protocols

Assay of cytokines in tissue culture supernatants describes a liquid suspension array for quantification of cytokines in tissue culture supernatants or serum. With this assay, it is possible to profile the level of multiple cytokines in a single well. The principle of this cytokine assay is similar to a capture sandwich immunoassay. Includes: Preparation for the Assay, Cytokine Assay, Reagents and Materials. - [Read Assay of Cytokines in Tissue Culture Supernatants]

BAC-FISH Protocol - http://www.nhm.ac.uk/hosted_sites/schisto/protocols/BAC_FISH.html

Category: BACs Bacterial Artificial Chromosome Protocols

Protocol uses the BIOPRIME reaction kit from GibcoBRL to prepare biotin-labelled BAC DNA which is detected using FITC-Avidin (Vector Labs, DCS grade). Reagents from other manufacturers may work equally well but have not been tested. Includes: Labeling of BAC clones; Ethanol precipitation; Hybridization; Post-hybridisation treatment / detection. - [Read BAC-FISH Protocol]

Binding Antibodies to Tissue Sections Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4331

Category: Antibody Protocols: Antibody Labeling Protocols

Once tissues are fixed and permeabilized, the antibodies are added. These antibodies can be labeled directly or detected by a labeled secondary reagent. For indirect detection, any reagent that binds specifically to the primary antibody can be "tagged" and used to locate the antibody. The possible reagents include anti-immunoglobulin antibodies, protein A or G, or, if the first antibody is labeled with biotin, streptavidin. They can be labeled with enzymes or gold. - [Read Binding Antibodies to Tissue Sections Protocol]

Cell Disruption and Subcellular Fractionation Protocol - http://www.duke.edu/web/ceramide/protocols/0011.html

Category: Cell Organelle Protocols: Subcellular Fractionation Protocols

Protocol for cell disruption and subcellular fractionation. Includes: Nitrogen Bomb, Fractionation and Reagents. - [Read Cell Disruption and Subcellular Fractionation Protocol]

Chemokine Receptor Expression Assay by Flow Cytometry - http://ubik.microbiol.washington.edu/protocols/facs/chemokinereceptorfacs.htm

Category: Signalling Signal Transduction Protocols: Chemokine Protocols

Chemokine Receptor Expression Assay by Flow Cytometry. Mullins Lab. Reagents, Protocol and detailed procedure. - [Read Chemokine Receptor Expression Assay by Flow Cytometry]

Complete Microarray Chip Protocol - http://www.rbhrfcrc.qimr.edu.au/kidney/Pages/Microarray%20protocol.html

Category: DNA Microarray Protocols

Reagents & Equipment, Labelling, Purification, Hybridisation, Washing. KRL Kidney Research Dr.Healt - [Read Complete Microarray Chip Protocol]

Construction of cDNA Libraries in Eukaryotic Expression Vectors for Construction and Screening - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot4068

Category: DNA Protocols: Genomic DNA Library Protocols

This stage summarizes how to construct a cDNA library in a mammalian vector, using commercial kits. These kits are available from several manufacturers and generally include all the required reagents. - [Read Construction of cDNA Libraries in Eukaryotic Expression Vectors for Construction and Screening]

Cyanine Dye (Maleimide) Protein Labeling Protocol - http://www.signaling-gateway.org/data/cgi-bin/ProtocolFile.cgi/afcs_PP00000150.pdf?pid=PP00000150

Category: Fluorescent Dyes Protocols

Cyanine dye reagents are useful as fluorescent labels for proteins. This protocol has been designed to label the thiol group on cysteine using Cy3 or Cy5 minimal maleimide labeling dyes. - [Read Cyanine Dye (Maleimide) Protein Labeling Protocol]

DNA Ligation Protocol PDF - http://www.oup.co.uk/pdf/pas/9v1-7-9.pdf

Category: Molecular Biology Protocols: DNA Ligation Protocols

DNA ligation Method, Equipment and reagents, T4 DNA ligase. Gannon and Powell. Oxford Practical Approach Series. - [Read DNA Ligation Protocol PDF]

DNA Miniprep Isolation from Plants Protocol - http://www.bio.com/protocolstools/protocol.jhtml?id=p1079

Category: Plant Biology Protocols: Plant DNA RNA Isolation Protocols

The original maize DNA miniprep protocol is used extensively for many plant species and different tissues. This slightly modified version is acceptable for most DNA extractions. The procedure has the advantage of speed and its use of inexpensive reagents. - [Read DNA Miniprep Isolation from Plants Protocol]

ELISA Method - http://mycoplasmas.vm.iastate.edu/lab_site/methods/immunol/ELISA.html

Category: ELISA Protocols

Reagents, protocol and Conjugate Preparation. Minion Lab, College of Veterinary Medicine, Iowa State. - [Read ELISA Method]

Fluorescent Reagents for Live Cell Imaging and Their Introduction into Cells - http://www.cshprotocols.org/cgi/content/abstract/2006/29/pdb.ip20

Category: Microscopy Protocols: Fluorescence Microscopy Protocols

Most biological specimens are relatively transparent, so details of internal and intracellular morphology are difficult to image in untreated living specimens using simple bright-field techniques. Fluorescence microscopy offers greater advantages and possibilities for increasing contrast and determining the specific localization of molecules in cells. Article outlines the three methods most commonly used to introduce an appropriate label into Drosophila tissue without perturbing the process. - [Read Fluorescent Reagents for Live Cell Imaging and Their Introduction into Cells]

Genetic Engineering with PCR Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3836

Category: PCR Protocols

Method describes how to modify the termini of PCR products by introducing restriction sites and other features. To reduce the chance of contamination with exogenous DNAs, prepare and use a special set of reagents and solutions for PCR only. Bake all glassware for 6 hours at 150°C and autoclave all plasticware. - [Read Genetic Engineering with PCR Protocol]

Genetic Engineering with PCR Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3836

Category: PCR Protocols: PCR Optimization

Immunoblotting: Antigen Detection Using Chemiluminescence Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/16/pdb.prot4271

Category: Western Blot Protocols: Antigen Detection Methods Protocols

The blot is blocked to prevent nonspecific adsorption of the immunological reagents. Antibodies are then bound to the proteins immobilized on the membrane, and the antigen is detected by labeling the antibodies with conveniently identified tags. Common labeling methods for chemiluminescent detection include anti-immunoglobulin antibody-coupled enzymes such as horseradish peroxidase, which catalyzes the oxidation of luminol and in turn releases light. - [Read Immunoblotting: Antigen Detection Using Chemiluminescence Protocol]

Immunoblotting: Antigen Detection Using Chromogenic Methods Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/16/pdb.prot4272

Category: Western Blot Protocols: Antigen Detection Methods Protocols

Immunofluorescence Reagents and Protocols - http://www.molbio1.princeton.edu/facility/flowcyt/if1.html

Category: Molecular Imaging: Immunofluorescence Microscopy

Immunofluorescence Reagents and Protocols. Staining Protocols. Princeton. - [Read Immunofluorescence Reagents and Protocols]

Long PCR Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3841

Category: PCR Protocols: Standard PCR Protocol

Protocol can be used to amplify DNA up to 25 kb in length. To reduce the chance of contamination with exogenous DNAs, prepare and use a special set of reagents and solutions for PCR only. Bake all glassware for 6 hours at 150°C and autoclave all plasticware. - [Read Long PCR Protocol]

Articles (1-6 of 6)

Single Stranded Plasmid DNA Isolation Protocol

A Single Stranded Plasmid DNA Isolation Protocol describing the production and isolation of single-stranded DNA (ssDNA) using bacteriophagemid-containing bacteria and helper phage. Infection of the host cells with helper phage allows for packaging of ssDNA into bacteriophage. The ssDNA can then be isolated from phage particles.

Acid Washing Microinjection Pipettes Protocol

Acid Washing of Glass microinjection pipettes protocol.

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.

Histone H1 Kinase Activity Assay

Histone H1 Kinase Activity Assay Protocol. This protocol describes assaying kinase activity of a putative kinase using Histone H1 as the substrate. Histone H1 is the canonical kinase substrate in this type of assay. Phosphorylation of Histone H1 is assessed by SDS-Polyacrylamide gel electrophoresis followed by autoradiography.

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.

Electrotransformation of BMH 81-17mut S for Isolating Site-Directed dsDNA Mutants

This protocol describes the electroporation of the BMH 81-17 mut S strain that is recommended for tranformation of the site directed mutagenesis of dsDNA (See Protocol on Site-Directed Mutagenesis on Double Stranded DNA). BMH 81-17 mut S are a mismatch repair defective (mut S) Escherichia coli strain. The probability that the two mutations will cosegregate during the first round of DNA replication is increased in this strain.

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