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ABH (p-Azidobenzoyl hydrazide) PDF - http://www.piercenet.com/files/0248dh4.pdf

Category: Molecular Biology Protocols: Carbohydrate Protocols

The ABH crosslinker binds to the Fc portion of an antibody molecule, away from the antigen binding site, resulting in a divalent immunologically active immunoglobulin derivative. Pierce - [Read ABH (p-Azidobenzoyl hydrazide) PDF]

Adenovirus Protocol - http://www.mc.vanderbilt.edu/root/vumc.php?site=mgcore&doc=4301

Category: Viral Culture and Isolation Protocols: Viral Isolation Protocols

Protocol for Adenovirus. - [Read Adenovirus Protocol]

Attaching Adaptors to Protruding Termini Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3920

Category: Cloning Protocols

Adaptors are short double-stranded synthetic oligonucleotides that carry an internal restriction endonuclease recognition site and single-stranded tails at one or both ends. Adaptors are used to exchange restriction sites at the termini of linear DNA molecules. They may be purchased in phosphorylated and unphosphorylated forms. - [Read Attaching Adaptors to Protruding Termini Protocol]

Colony PCR Protocol - http://www.enterococcus.ouhsc.edu/ColonyPCRProtocol.asp

Category: PCR Protocols: Colony PCR

PCR Reaction Conditions for Colony PCR. Contributed by Lynn Hancock. Enterococcus Research Site, The Board of Regents of the University of Oklahoma. - [Read Colony PCR Protocol]

Colony PCR Protocol II - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4141

Category: PCR Protocols: Colony PCR

Protocol describes the use of PCR to screen for bacteria that carry recombinant plasmids. The PCR can be carried out using the same primers as for amplification of the cloned insert. To determine the orientation of the insert, a third, insert-specific primer that is asymmetrically distanced from the clonal insertion site can be used. - [Read Colony PCR Protocol II]

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]

Cryo-Immunogold Electron Microscopy Protocol - https://catalog.invitrogen.com/index.cfm?fuseaction=iProtocol.unitSectionTree&treeNodeID=9E662E9BAD69DF0691FE8955F6EE7024&objectid=6673D13FDA2B0D70D4143DB7CECC80D9

Category: Molecular Imaging: Electron Microscopy Protocols

Details of this protocol, Cryo-Immunogold Electron Microscopy, are located on a web site other than Protocols. Cryo-Immunogold Electron Microscopy Protocol - [Read Cryo-Immunogold Electron Microscopy Protocol]

DNA Affinity Chromatography Using Gravity Flow - Subscription Required - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4206

Category: Chromatography Protocols: DNA RNA Affinity Chromatography

DNA Affinity Chromatography, DNA affinity chromatography can be a low-tech method using gravity flow at 4Â°C, a disposable chromatography column, and DNA affinity resin prepared in the laboratory (see Preparation of a DNA Affinity Column). Include 10-20% glycerol and 0.025-0.1% NP-40 in the column buffers to suppress losses due to nonspecific adsorption of protein to surfaces. Load the protein in a buffer that is compatible with binding of the protein to its target site. Keith Brocklehurst et al - [Read DNA Affinity Chromatography Using Gravity Flow - Subscription Required]

DNA In Situ Hybridization with an Alkaline Phosphatase-Based Fluorescent Detection System - http://www.roche-applied-science.com/PROD_INF/MANUALS/InSitu/pdf/ISH_124-126.pdf

Category: Cytogenetics Protocols: DIG Biotin Labeled Probe Protocols

Protocol describes here a high sensitivity indirect detection procedure for DIG-labeled hybridization probes. The procedure uses the components of the HNPP Fluorescent Detection Set to form a fluorescent precipitate of HNPP (2-hydroxy-3-naphthoic acid-2’-phenylanilide phosphate) and Fast Red TR at the site of hybridization. Includes: In situ hybridization with DIG-labeled probes; Detection of DIG-labeled probes; Fluorescence microscopy. - [Read DNA In Situ Hybridization with an Alkaline Phosphatase-Based Fluorescent Detection System]

DNA In Situ Hybridization with an Alkaline Phosphatase-Based Fluorescent Detection System Protocol - http://www.roche-applied-science.com/PROD_INF/MANUALS/InSitu/pdf/ISH_124-126.pdf

Category: Nucleic Acid Modification Protocols: DIG Biotin Labeled Probes Protocols

Protocol describes a high sensitivity indirect detection procedure for DIG-labeled hybridization probes. The procedure uses the components of the HNPP Fluorescent Detection Set to form a fluorescent precipitate of HNPP (2-hydroxy-3-naphthoic acid-2’-phenylanilide phosphate) and Fast Red TR at the site of hybridization. This procedure can be used to detect single copy sequences as small as 1 kb on human metaphase chromosomes. - [Read DNA In Situ Hybridization with an Alkaline Phosphatase-Based Fluorescent Detection System Protocol]

Establishment of Stable Transfectant of CHO Lec Cells Protocol - http://www.cbrinstitute.org/labs/springer/protocols/jun_CHOlec.html

Category: Transfection Protocols: Stable Transfection Protocols

CHO Lec3.2.8.1 cells have 4 independent mutations in the N and O glycosylation pathways. When cultured with alpha-glucosidase I inhibitor N-butyl-deoxynojirimycin, glycoproteins produced in CHO Lec3.2.8.1 cells are completely susceptible to Endo H digestion. Endo H cleaves chitobiose, leaving a single N-linked N-acetylglucosamine per site which is ideal for maintenance of protein solubility and special carb-protein interactions, such as between the first N-acetyl glucosamine residue and tryp. - [Read Establishment of Stable Transfectant of CHO Lec Cells Protocol]

Functional Analyses for Site-Specific Phosphorylation of a Target Protein in Cells - http://www.natureprotocols.com/2007/01/10/functional_analyses_for_sitesp.php

Category: Antibody Protocols: Antibody Production Protocols

Novel strategy of immunizing a phosphorylated peptide or a synthetic phosphopeptide, which corresponds to the protein phosphorylated at a targeted residue. Method has been applied to the production of antibodies that can specifically recognize the other types of site-specific protein modification, such as acetylation, methylation, and proteolysis. - [Read Functional Analyses for Site-Specific Phosphorylation of a Target Protein in Cells]

Generation of Sets of Nested Deletion Mutants with Exonuclease III Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot4078

Category: Genetics and Genomics: Genotyping Protocols: Mutation Detection Protocols

The double-stranded DNA of recombinant plasmid, phagemid, or bacteriophage M13 replicative form DNA is digested with two restriction enzymes whose sites of cleavage both lie between one end of the target DNA and the binding site for universal primer. The enzyme that cleaves nearer the target sequence must generate either a blunt end or a recessed 3' terminus; the other enzyme must generate a four-nucleotide protruding 3' terminus. - [Read Generation of Sets of Nested Deletion Mutants with Exonuclease III Protocol]

Introduction of Double-Stranded RNA in C. elegans by Injection Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4315

Category: C. elegans Protocols: C. elegans Genetics Protocols

Double-stranded RNA (dsRNA) can be efficiently introduced into Caenorhabditis elegans by microinjection into the gonad, the gut, or the body fluid. The RNAi effect will spread within the nematode, exerting an effect beyond the site of injection. - [Read Introduction of Double-Stranded RNA in C. elegans by Injection Protocol]

Introduction of Double-Stranded RNA in C. elegans by Injection Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4315

Category: RNAi Technology Protocols: RNAi C. elegans Protocols

Isolating the Ends of Genomic DNA Fragments Cloned in High-capacity Vectors - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot4009

Category: PCR Protocols: PCR Cloning Protocols

Protocol describes the use of vectorette PCR and single-site PCR to amplify the terminal sequences of genomic sequences cloned in high-capacity vectors such as PACs and YACs. - [Read Isolating the Ends of Genomic DNA Fragments Cloned in High-capacity Vectors]

Large-scale Preparation of Single-stranded and Double-stranded Bacteriophage M13 DNA Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3284

Category: Viral Manipulation Protocols: M13 Phage Manipulation Protocols

Protocol used chiefly to generate large stocks of double-stranded DNA of strains of M13 that are routinely used as cloning vectors. Large amounts of single-stranded DNA of an individual recombinant may occasionally be needed for specific purposes, e.g., to generate many preparations of a particular radiolabeled probe or to construct large numbers of site-directed mutants. - [Read Large-scale Preparation of Single-stranded and Double-stranded Bacteriophage M13 DNA Protocol]

Oligonucleotide-directed Mutagenesis by Elimination of a Unique Restriction Site (USE Mutagenesis) - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3742

Category: Cloning Protocols: Mutagenesis Protocols

Protocol for oligonucleotide-directed mutagenesis by elimination of a unique restriction site (USE mutagenesis). - [Read Oligonucleotide-directed Mutagenesis by Elimination of a Unique Restriction Site (USE Mutagenesis)]

Photoactivation-Based Labeling and In Vivo Tracking of RNA Molecules in the Nucleus - http://www.cshprotocols.org/cgi/content/abstract/2006/29/pdb.prot4600

Category: Microscopy Protocols: Live-Cell Imaging Protocols

This protocol describes a method for observing and measuring the movement of RNA molecules in the nucleus of living mammalian cells. Caged fluorescein-labeled DNA oligonucleotides are introduced into living mammalian cells, where they demonstrably hybridize to complementary RNA. After site-specific photoactivation at desired sites within the cell, the RNA movements away from those sites are followed and digitally recorded using a rapid acquisition microscopy system. - [Read Photoactivation-Based Labeling and In Vivo Tracking of RNA Molecules in the Nucleus]

Purification of Nuclear Factor DNA Recognition Site Affinity PDF - http://www.jbc.org/cgi/reprint/261/3/1398.pdf

Category: Chromatography Protocols

Purification of Nuclear Factor DNA Recognition Site Affinity. PDF. - [Read Purification of Nuclear Factor DNA Recognition Site Affinity PDF]

Rapid and Efficient Site-directed Mutagenesis by the Single-tube Megaprimer PCR Method Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3467

Category: Cloning Protocols: Mutagenesis Protocols

Protocol for rapid and efficient site-directed mutagenesis by the single-tube megaprimer PCR method. - [Read Rapid and Efficient Site-directed Mutagenesis by the Single-tube Megaprimer PCR Method Protocol]

Rapid PCR Site-Directed Mutagenesis Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4144

Category: Cloning Protocols: Mutagenesis Protocols

Protocol for rapid PCR site-directed mutagenesis. - [Read Rapid PCR Site-Directed Mutagenesis Protocol]

Screening Recombinant Clones for Site-directed Mutagenesis by Hybridization to Radiolabeled Oligos - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot4013

Category: Viral Manipulation Protocols: M13 Phage Manipulation Protocols

Plaques formed by M13 bacteriophages or bacterial colonies transformed by plasmids carrying specific mutations can be detected by hybridization, using a radiolabeled oligonucleotide that forms a perfect duplex with the mutant sequence. Hybridization is carried out under conditions of low stringency that allow the radiolabeled oligonucleotide to anneal to both mutant and wild-type DNAs. - [Read Screening Recombinant Clones for Site-directed Mutagenesis by Hybridization to Radiolabeled Oligos]

Singel Nucleotide Primer Extension (SNuPE) - http://www.methods.info/Methods/DNA_methylation/SNuPE.html

Category: Epigenetics and Methylation Protocols: Bisulfite Treatment Methylation Assay Protocol

Single Nucleotide Primer Extension can be used for the analysis of methylation in a certain position. Treat DNA with bisulphite and then anneal a primer which ends immediately before the site of analysis. Dr. A. Gratchev Methods.info - [Read Singel Nucleotide Primer Extension (SNuPE)]

Site Directed Mutagenesis Protocol - http://openwetware.org/wiki/Knight:Site-directed_mutagenesis

Category: Cloning Protocols: Mutagenesis Protocols

Protocol for site directed mutagenesis. - [Read Site Directed Mutagenesis Protocol]

Articles (1-5 of 5)

Lambda Phage Protocol Large DNA Preparation

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.

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.

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.

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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