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3' Rapid Amplification of cDNA Ends (RACE) Protocol - http://www.nottingham.ac.uk/~mbzspd/methods/3RACE_PCR.html

Category: RNA Protocols: 3' RACE Rapid Amplification of cDNA Ends Protocols

PCR cycle steps, protocol using SuperScript II reverse transcriptase (Life Technologies) and Taq polymerase, Gene specific primer, T17 Adapter primer and an Adapter primer. Dr.Dawson, Nottingham. - [Read 3' Rapid Amplification of cDNA Ends (RACE) Protocol]

AFLP PCR Protocol PDF - http://www4.ncsu.edu/~wagebrey/AFLP-Salm.pdf

Category: PCR Protocols: AFLP PCR

Restriction digestion, Adapter ligation, Preamplification, Final Amplification steps for AFLP PCR. Detailed protocol. Wondwossen Abebe Gebreyes - [Read AFLP PCR Protocol PDF]

Analysis of Flow Cytometry Data Protocol - https://catalog.invitrogen.com/index.cfm?fuseaction=iProtocol.unitSectionTree&treeNodeID=9E661C2CE22FCCB1C294CD8376FD8830&objectid=6674E762AC837B13929440A1F32AAEF0

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: Flow Cytometry Data Analysis Protocols

Provides several approaches to flow cytometry data analysis. Frequency determinations based on analysis of single-parameter fluorescence histograms and dual-parameter contour plots are presented. Steps are described for calculating values for signal-to-noise ratios when logarithmic amplification is used for data collection. - [Read Analysis of Flow Cytometry Data Protocol]

Basic ELISA Method - http://iacf.bsd.uchicago.edu/Monoclonal_Antibody_Facility/BasicELISAProtocol.htm

Category: ELISA Protocols

Basic ELISA Method. All the steps for the basic ELISA protocol. Immunology Applications Core Facility, Univ. Chicago. - [Read Basic ELISA Method]

Bradford Protein Assay - http://www.ruf.rice.edu/~bioslabs/methods/protein/bradford.html

Category: Protein Protocols: Protein Quantitation Concentration Protocols: Bradford Protein Assay Protocols

A detailed and well thought out Bradford protein assay page using a spectrophotometer. Includes information such as the Bradford assay is very fast and uses about the same amount of protein as the Lowry assay, comments, procedural steps, equipment used and more. - [Read Bradford Protein Assay]

Bradford Protein Concentration Assay - http://web.chemistry.gatech.edu/~williams/bCourse_Information/4581/techniques/bradford/bradford.html

Category: Protein Protocols: Protein Quantitation Concentration Protocols: Bradford Protein Assay Protocols

Includes Abbreviations, Background, and Procedure steps using BSA. The Bradford protein assay (1) is one of several simple methods commonly used to determine the total protein concentration of a sample. The method is based on the proportional binding of the dye Coomassie to proteins. The assay is colorimetric; as the protein concentration increases, the color of the test sample becomes darker. Coomassie absorbs at 595 nm. - [Read Bradford Protein Concentration Assay]

Chromatin Immunoprecipitation Protocol - http://www.microarrays.ca/support/PDFs/ChIP_Protocol_for_Microarray_Analysis_Staph_A_Cells_09-07-04.pdf

Category: DNA Protocols: Chromatin Immunoprecipitation Protocols

ChIP Staph A cell preparation, preblocking and ChIP IP preclear, recovery, wash steps based on Farnham Lab Protocols. - [Read Chromatin Immunoprecipitation Protocol]

Chromatin IP (CHIP assay) - http://www.fhcrc.org/science/labs/breeden/Methods/chromatinIP.html

Category: DNA Protocols: Chromatin Immunoprecipitation Protocols

ChIP assay protocol with 2 steps: in vivo formaldehyde cross-linking of whole cells and protein-protein and protein-DNA interactions, followed by immunoprecipitation of protein-DNA complexes with specific antibodies from sonicated extracts. Breeden Lab - [Read Chromatin IP (CHIP assay)]

Construction of Genomic DNA Libraries in Cosmid Vectors Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot4000

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]

Cryosectioning Protocol - http://genetics.med.harvard.edu/~cepko/protocol/mike/C7.html

Category: Immunohistochemistry Protocols: Cryosectioning Protocols

Protocol for cryosectioning. While the timing of the various steps in this protocol are probably not critical, process the tissue all at once to ensure that RNA and/or proteins do not get degraded. Includes: 20% Paraformaldehyde/4% Paraformaldehyde-PBS; Sucrose/PBS. - [Read Cryosectioning Protocol]

Deglycosylation of Glycoproteins Using Endoglycosidases - http://www.glycotech.com/protocols/Proto3.html

Category: Molecular Biology Protocols: Carbohydrate Protocols

Deglycosylation steps are different with every glycoprotein and must be determined empirically (Methods in Enzymology, 1994, 230: 44-57). A typical test reaction of human transferrin is described. T.H. Plummer, Jr. and A.L. Tarentino, Department of Biochemistry, New York State Department of Health, Wadsworth Center for Laboratories and Research, Albany, New York - [Read Deglycosylation of Glycoproteins Using Endoglycosidases]

DNA Ligation - http://www.bio.com/protocolstools/protocol.jhtml?id=p2053

Category: Molecular Biology Protocols: DNA Ligation Protocols

Description and protocol of the steps required to join together plasmid and insert fragments to create a new plasmid. Paul Kaufman, Univ California. - [Read DNA Ligation]

Fabrication Protocol for DNA Microarrays - http://research.nhgri.nih.gov/microarray/fabrication.shtml

Category: DNA Microarray Protocols

This protocol describes the steps required to produce a cDNA microarray. Gene-specific DNA is produced by PCR amplification of purified template plasmid DNAs from cloned ESTs. The PCR product is purified by ethanol precipitation, thoroughly resuspended in - [Read Fabrication Protocol for DNA Microarrays]

First strand cDNA synthesis with Ready-To-Go Beads Protocol - http://www.methods.info/Methods/cDNA/cDNA_ready_to_go.html

Category: PCR Protocols: cDNA Synthesis Protocols

This cDNA synthesis system simplifies your work dramatically. All reaction components are premixed and lyophylised. You have to add your RNA and (for Your-Prime beads) the primer. Another advantage of the system is a little number of pipetting steps required, and therefore reduced risk of Rnase contamination and RNA degradation. - [Read First strand cDNA synthesis with Ready-To-Go Beads Protocol]

Fungal DNA Isolation Method - http://wheat.pw.usda.gov/~lazo/methods/lazo/dnafun.html

Category: DNA Protocols: DNA Extraction Protocols

DNA isolation from various fungal species including: Cochliobolus, Aternaria, and Fusarium. Key steps: (1) the use of young lyophilized mycelial mats - yield less contaminating carbohydrates; (2) proteinase K in the extraction buffer to destroy DNases (f - [Read Fungal DNA Isolation Method]

How to do a Western Blot - http://www.well.ox.ac.uk/flint/Western.htm

Category: Protein Protocols: Western Blot Protocol

All the products and steps to complete a full western blotting analysis. (Jonathan Flint lab). - [Read How to do a Western Blot]

Method: Preparation of Lymphocyte Cell Pellet for Storage - http://humgen.wustl.edu/hdk_lab_manual/hcc/hcc5.html

Category: Cell Biology and Cell Culture: Cell Culture Techniques: Cell Line Preservation Freezing

Following propagation to 1 X 108 cells, lymphoblastoid cells are conveniently stored at -80 degrees C to preserve the high molecular weight DNA in the cells until the DNA is purified. This procedure describes the steps required to harvest and freeze the c - [Read Method: Preparation of Lymphocyte Cell Pellet for Storage]

MTT Cell Proliferation Assay Protocol - http://www.atcc.org/pdf/30-1010k.pdf

Category: Cell Biology and Cell Culture: Cell Viability Protocols

The MTT Cell Proliferation Assay measures the cell proliferation rate and conversely, when metabolic events lead to apoptosis or necrosis, the reduction in cell viability. The number of assay steps has been minimized as much as possible to expedite sample processing. The MTT Reagent yields low background absorbance values in the absence of cells. Includes: Determining optimal cell counts, performing an assay, data interpretations and troubleshooting. - [Read MTT Cell Proliferation Assay Protocol]

Neural Stem Cell Culture: Neurosphere Generation, Microscopical Analysis and Cryopreservation - http://www.natureprotocols.com/2006/08/25/neural_stem_cell_culture_neuro.php

Category: Stem Cell Protocols: Stem Cell Culture Protocols

Describes the steps in detail to isolate and expand neural stem cells in the form of neurospheres from tissue dissections of the post-natal mouse brain. Procedures for the long term passage of neurospheres and the cryopreservation of neurospheres are also provided. In addition to the guidelines and tips for generating neurosphere cultures, we describe the method to prepare neurospheres for analysis by light microscopy. - [Read Neural Stem Cell Culture: Neurosphere Generation, Microscopical Analysis and Cryopreservation]

Precision Engineering of Plant Gene Loci by Homologous Recombination Cloning in Escherichia Coli - http://www.plantmethods.com/content/1/1/6

Category: Plant Biology Protocols: Arabidopsis Genetics Protocols

Protocol for precision engineering of plant gene loci by homologous recombination cloning in Escherichia coli. Includes: Key steps in the EL250 RED-HR locus rescue and engineering procedure; Primer design and plasmid constructs; AtSTM gap-repair construct; Targeting construct backbone; Preparation of electrocompetent EL250 cells; Transformation of BAC F24o1 and induction of recombinogenic function in EL250; AtSTM locus rescue from BAC F24o1 by gap-repair HR. - [Read Precision Engineering of Plant Gene Loci by Homologous Recombination Cloning in Escherichia Coli]

Preparation and Transformation of Competent E. coli Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3942

Category: E Coli Protocols: E coli Transformation Protocols

Procedure generates competent cultures of E. coli that can be transformed at high frequencies (5 x 108 transformed colonies/µg of superhelical plasmid DNA). IMPORTANT All steps in this protocol should be carried out aseptically. - [Read Preparation and Transformation of Competent E. coli Protocol]

Preparing Chemically Competent Cells - http://openwetware.org/wiki/Preparing_chemically_competent_cells

Category: Microbiology: Bacterial Competent Cells Preparation E.Coli

Detailed protocol Preparation and Steps for E.Coli Competent Cells procedure. Open Net Ware. - [Read Preparing Chemically Competent Cells]

Reverse Transcriptase In Situ PCR Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/14/pdb.prot4119

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]

Reverse Transcriptase In Situ PCR Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/14/pdb.prot4119

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]

RT In Situ PCR - Foundation of Success - http://www.bioscience.org/1996/v1/c/nuovo1/htmls/list.htm

Category: PCR Protocols: In-situ PCR Protocols

The key preparatory steps. In-situ RT-PCR. Fixative. Protease Digestion. DNase digestion. One-step in-situ RT PCR Protocol. Gerard J. Nuovo - [Read RT In Situ PCR - Foundation of Success]

Articles (1-7 of 7)

Tubulin Polymerization Protocol using GTP and GMPCPP

Tubulin is polymerized into microtubules by incubating tubulin at 37°C with GTP. A nucleation seed is added when the purpose is to assay microtubule elongation. Tubulin can also be polymerized for the purposes of recycling the tubulin or labeling the microtubules with fluorescently labeled tubulin. Based on the protocol by Timothy Mitchison of Harvard University.

Paraffin Embedding Protocol

Paraffin Embedding Protocol for molecular profiling. This Paraffin Embedding Protocol describes the processing of the tissues into sections following ethanol fixation. Molecular profiling (MP) is a technique that is used to visualize the global patterns of RNA expression or protein expression in various cell types and disease processes.

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.

Immobilized Antigen Phage Antibody Selection Protocol

A protocol for the selection of Phage Antibodies using Immobilized Antigen. This method describes the selection of antibodies from bacteriophage antibody libraries that recognize a specific antigen. The phage display library of antibody-displaying phage particles is exposed to antigen attached to a solid substrate (Nunc Immuno™ tubes). The phage particles with affinity for antigen bind to the immobilized antigen and are selected from the library of phage expressing antibodies.

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.

DNA Ligation Protocol

The DNA Ligation protocol described here contains the steps required to join together using ligase enzyme both plasmid DNA and insert DNA fragments in order to create a new plasmid. This new ligated plasmid can be transformed after into competent bacteria to produce DNA for mini, midi or maxi-prep isolation.

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.

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