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2-D Polyacrylamide Gel Electrophoresis Protocol - http://cgap-mf.nih.gov/Protocols/DNARNAProteomicAnalysis/Proteomics/2DPage.html

Category: Primary Cell Isolation and Culture Protocols: Laser Capture Microdissection Protocols

Protocol for 2-D polyacrylamide gel electrophoresis. Includes: 50 ml IEF Lysis Buffer; 10X Electrophoresis Running Buffer (10 L); 30% Acrylamide Stock (1 liter); Separating Acrylamide Gel; 50 ml Equilibration Buffer I; 50 ml Equilibration Buffer II; Transfer Buffer; Sample Preparation; Tissue Processing; Reswelling; Prepare Gradient Acrylamide Gel (9-18%); Transfer and Sequencing of Proteins. - [Read 2-D Polyacrylamide Gel Electrophoresis Protocol]

96-Well Confirmation Yeast PCR Protocol - http://sequence-www.stanford.edu/group/yeast_deletion_project/96_well_confirmation.html

Category: Yeast Protocols: Yeast Nucleic Acid Protocols: Yeast PCR Protocols

Protocol for 96-well confirmation Yeast PCR. Includes: Clonal purification; Generate a master plate (96-well format); Making a frozen backup stock; Confirmation PCR for one Row; ORF Specific Confirmation PCR --> "A-B" primers (upstream junction); Transfer template DNA to multiwell PCR plate; Prepare and dispense master mix for A-B PCR. - [Read 96-Well Confirmation Yeast PCR Protocol]

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]

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]

Bioresponsive Targeted Charge Neutral Lipid Vesicles for Systemic Gene Delivery Protocol - http://www.cshprotocols.org/cgi/content/short/2006/1/pdb.prot4450

Category: Gene Delivery Protocols: DNA Nanoparticles Protocols

This protocol describes a stepwise procedure to prepare nucleic acids encapsulated in a polyethylene glycol (PEG)-shielded nanolipoparticle (NLP) that contain a bioresponsive lipid and ligand. This process provides several advantages for systemic gene delivery. The in vivo circulation time is extended. Also, low pH-sensitive lipids enhance DNA unpacking and endosomal escape. Finally, ligands inserted into the NLP surface can target gene delivery to specific tissues or cells in vivo. - [Read Bioresponsive Targeted Charge Neutral Lipid Vesicles for Systemic Gene Delivery Protocol]

Cell Lysates For Western Blotting - http://www.liv.ac.uk/~giles/methods/western_lysate.htm

Category: Protein Protocols: Western Blot Protocol

How to prepare cell lysates for western blotting. - [Read Cell Lysates For Western Blotting]

ChIP TIPS PDF - http://www.upstate.com/img/pdf/ChIP_Protocol_thin_v2.pdf

Category: DNA Protocols: Chromatin Immunoprecipitation Protocols

Prepare Chromatin, Immunoprecipitate, Wash, Elute, IP Procedure. UpState. - [Read ChIP TIPS PDF]

Clone Genes From a Phage Library Protocol - http://www.ucsf.edu/micro/faculty/Johnson/protocols/protocol1.html

Category: DNA Protocols: cDNA Library Protocols: cDNA Library Construction Protocols

Protocol for cloning genes from a phage library. Includes: Titer and plate out phage; Lift plaques onto filters and prepare them for screening; Make a probe; Hybridize the probe to the filters; Wash the filters and expose to film; Purify putative plaques; Excise plasmid from the desired phage. - [Read Clone Genes From a Phage Library Protocol]

Construction of a Yeast Genomic Library Protocol - http://www.bio.com/protocolstools/protocol.jhtml?id=p361

Category: DNA Protocols: Genomic DNA Library Protocols

Protocol for the construction of a Yeast genomic library. Includes: Prepare the genomic DNA; Prepare the Library Vector; Ligate the Digested Genomic DNA to the Digested Vector DNA; Prepare Library DNA from Bacteria. - [Read Construction of a Yeast Genomic Library Protocol]

Denaturing Agarose Gel Electrophoresis of RNA Protocol - http://www.ambion.com/techlib/append/supp/rna_gel.html

Category: Nucleic Acid Electrophoresis Protocols: RNA Electrophoresis Protocols

Protocol for denaturing agarose gel electrophoresis of RNA. Includes: Prepare the gel; Prepare the RNA sample; Electrophoresis. - [Read Denaturing Agarose Gel Electrophoresis of RNA Protocol]

Expression Protocol in M9 Minimal Media via T7 Promoter - http://structbio.vanderbilt.edu/chazin/wisdom/labpro/M9.html

Category: Microbiology: Bacterial E.Coli Culture Media & Plates

How to prepare M9 minimal media and grow in M9 media. Chazin Lab , Vanderbilt Univ. - [Read Expression Protocol in M9 Minimal Media via T7 Promoter]

FAQs About Cell Sorting - http://cyto.mednet.ucla.edu/Protocols/question.htm

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

Questions and answers about cell sorting. Includes: When should I use fluorescence activated cell sorting over bulk separation methods like panning or magnetic bead separations? Will my cells be harmed by the sorting process? How many cells do I need to prepare to recover 1 X 106 of a population that comprises 10% of the cells? Are there ways to improve sort recovery? etc... - [Read FAQs About Cell Sorting]

Frozen Yeast Transformation Protocol - http://www.umanitoba.ca/faculties/medicine/biochem/gietz/Frozen.html

Category: Yeast Protocols

This Protocol allows You to prepare Frozen Yeast cells that are competent for transformation after thawing. - [Read Frozen Yeast Transformation Protocol]

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

Growth and Concentration of Chlorogonium for Culturing Freshwater Hypotrichs Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/21/pdb.prot4464

Category: Laboratory Model Organisms Protocols

Protocol describes the growth and concentration of the alga Chlorogonium elongatum as a food source for culturing freshwater hypotrichs. Most freshwater hypotrichs (including Oxytricha nova, O. fallax, and O. trifallax; Euplotes aediculatus and E. eurystomous; and Stylonychia lemnae) can be grown to high density with Chlorogonium as the food organism. A similar regimen can be used to prepare other food sources such as Tetrahymena or bacteria (e.g., Aerobacter aerogenes). - [Read Growth and Concentration of Chlorogonium for Culturing Freshwater Hypotrichs Protocol]

How to Choose and Prepare Media - http://www.fgsc.net/neurosporaprotocols/How%20to%20choose%20and%20prepare%20media.pdf

Category: Fungi Protocols: Neurospora Protocols

Information and tips on how to choose and prepare media. - [Read How to Choose and Prepare Media]

How to Prepare Aceto-Orcein Squashes, Especially for Pachytene Chromosomes - http://www.fgsc.net/neurosporaprotocols/How%20to%20prepare%20aceto-orcein%20squashes,%20especially%20for%20pachytene%20chro.pdf

Category: Fungi Protocols: Neurospora Protocols

Information and tips on how to prepare aceto-orcein squashes, especially for pachytene chromosomes. - [Read How to Prepare Aceto-Orcein Squashes, Especially for Pachytene Chromosomes]

How to Prepare and Use Gradient Plates - http://www.fgsc.net/neurosporaprotocols/How%20to%20prepare%20and%20use%20gradient%20plates.pdf

Category: Fungi Protocols: Neurospora Protocols

Information and tips on how to prepare and use gradient plates. - [Read How to Prepare and Use Gradient Plates]

How to Prepare Ascus Rosettes for Microscopic Examination - http://www.fgsc.net/neurosporaprotocols/How_to_obtain_rosettes%20final.pdf

Category: Fungi Protocols: Neurospora Protocols

Information and tips on how to prepare ascus rosettes for microscopic examination. - [Read How to Prepare Ascus Rosettes for Microscopic Examination]

Isolation of DNA from Mammalian Cells by Spooling Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot4037

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols: DNA Isolation from Mammalian Cells Protocols

Procedure is used to prepare DNA simultaneously from many different types of samples or tissues. Although the DNA is generally too small (approx. 80 kb) for efficient construction of genomic DNA libraries, it gives excellent results in Southern hybridizations and PCRs. Cultured aneuploid mammalian cells (2 x 107, e.g., HeLa cells) yield 100 µg of DNA in a volume of 1 ml. - [Read Isolation of DNA from Mammalian Cells by Spooling Protocol]

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]

Long Term Lambda Phage Storage Protocol - http://humgen.wustl.edu/hdk_lab_manual/lambda/lambda6.html

Category: Cell Culture Protocols: Cell Storing and Cell Freezing Protocols

No special treatment is required to prepare a lysate for the active collection. The following procedure should be used for long-term storage of lambda clones in the archival collections. The phage are diluted in media containing 7% DMSO and frozen at -80 degrees C. - [Read Long Term Lambda Phage Storage Protocol]

Lysis and Protein Extraction from WEHI-231 Cells with TriPure Isolation Reagent Protocol - http://www.signaling-gateway.org/data/cgi-bin/ProtocolFile.cgi/afcs_PP00000162.pdf?pid=PP00000162

Category: Chromatography Protocols: Immobilized Metal Ion Affinity Chromatography Protocols

Protocol is the first step in a three-step process for the preparation and enrichment of phosphopeptides using immobilized metal affinity chromatography (IMAC) for the identification of the phosphopeptides by LC-MS/MS. This procedure is used to prepare protein extracts from WEHI-231 cells. This preparation method provides total cellular protein samples that are free of contaminating nucleic acids. - [Read Lysis and Protein Extraction from WEHI-231 Cells with TriPure Isolation Reagent 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]

Articles (1-10 of 13)

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.

Microarray Protocol Preparation of Fluorescent DNA Probes from Human mRNA

This Microarray Protocol Preparation of Fluorescent DNA Probes from Human mRNA protocol describes the production of probes labeled with the fluorescent dyes, Cy3 and Cy5, following the synthesis of cDNA from human mRNA and the hybridization of the probes to DNA microarrays.

Probe Preparation for 22 x 40 mm DNA Microarrays

Probe Preparation for 22 x 40 mm DNA Microarrays Protocol.

Microinjection Pipette Pulling Protocol

A simple protocol for microinjection pipette pulling.

Antibodies Phage Expression Protocol Using 96-well Microtiter Plates

A Phage display expression protocol for antibody expression in 96-well microtiter plates.

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.

Coupling Cysteine Containing Peptides to Carrier Protein for Immunization and Polyclonal Antibody

This protocol is used in the production of polyclonal antibodies that recognize a peptide sequence of interest.

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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