enzyme Protocols

Category: DNA Protein Interactions Protocols: Dnase Footprinting Assay Protocols

Protocol describes how isolated nuclei are incubated with varying amounts of Dnase I. Genomic DNA is then isolated from the nuclei and digested with a restriction enzyme, analyzed by gel electrophoresis, and probed by Southern hybridization. - [Read Mapping Dnase-I-hypersensitive Sites Protocol]

Category: Cytogenetics Protocols

Protocol describes a recently developed method — methylation-specific digital karyotyping (MSDK) — that enables comprehensive and unbiased genome-wide DNA methylation analysis. Using a combination of a methylation-sensitive mapping enzyme (for example, AscI) and a fragmenting enzyme (for example, NlaIII), short sequence tags can be obtained and uniquely mapped to genome location. - [Read Methylation-Specific Digital Karyotyping Protocol]

Category: Molecular Biology Protocols: Restriction Enzyme Digestion

Molecular Cloning Lab – Restriction Enzyme Digestion. Molecular Cloning introduction, lab for students - restriction digest exercise with questions. BE209 Spring 2006. Prof. Timothy Gardner. Boston University, Department of Biomedical Engineering. - [Read Molecular Cloning Lab – Restriction Enzyme Digestion PDF]

Category: Cytogenetics Protocols: In Situ Hybridization Protocols

Protocol for multiple-target DNA in situ hybridization with enzyme-based cytochemical detection systems. Includes: Cell preparations; Cell processing; Probe preparation; Multiple-target in situ hybridization (ISH); ISH with separate probe and target denaturation [for probes with repetitive (e.g., Alu) elements]; Post-hybridization washes; Enzyme-based cytochemical detection; etc.. - [Read Multiple-Target DNA In Situ Hybridization with Enzyme-Based Cytochemical Detection Systems Protocol]

Category: Neuroscience Protocols: Histology Stains Protocols: Enzyme Histochemical Stains Protocols

Protocol stain for NADH diaphorase enzyme. - [Read NADH Diaphorase Enzyme Histochemical Stain Protocol]

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols: Genomic DNA Isolation Protocols

Protocol is used to establish conditions for restriction enzyme digestion of eukaryotic genomic DNA that will generate fragments of a size appropriate for construction of genomic libraries.  To construct a genomic library, the average length of the starting genomic DNA should be at least eight times the capacity of the vector. - [Read Partial Digestion of Eukaryotic DNA for Use in Genomic Libraries: Pilot Reactions Protocol]

Category: Plant Biology Protocols: Photosynthesis and Respiration Protocols

The physiological reactions of mitochondria and chloroplasts can be reduced to a series of electron transfers, catalyzed by specific enzymes found within the organelles. Thus, we can study the component processes of photosynthesis and respiration by isolating the organelles and measuring specific enzyme activity associated with that organelle. - [Read Photosynthesis and Respiration - Introduction]

Category: Signalling Signal Transduction Protocols

This protocol provides a sufficient sample for several determinations of cAMP using the acetylation protocol. The method chosen for measuring the content of cyclic adenosine 3',5'-monophosphate (cyclic AMP or cAMP) in splenic B lymphocytes (B cells) is an enzyme linked immunoassay system. Protocol includes information on: Treatment of Cells and Preparation of Extracts; Reagents and Materials. - [Read Preparation of B-Lymphocyte Lysates for Cyclic AMP Determination]

Category: Viral Manipulation Protocols: Phage Lamda Manipulation Protocols

Bacteriophage {lambda} vectors that allow genetic selection of recombinant bacteriophages (e.g., the EMBL series, {lambda}2001, {lambda}DASH, {lambda}ZAP, and {lambda}gt10) can be prepared for cloning by simple digestion by one or more restriction enzymes. - [Read Preparation of Bacteriophage lambda DNA Cleaved with a Single Restriction Enzyme Protocol]

Category: Nucleic Acid Electrophoresis Protocols: Pulsed Field Gel Electrophoresis Protocols

Protocol for preparation of DNA for pulsed-field gel electrophoresis: isolation of DNA from mammalian cells and tissues. Genomic DNAs from mammalian cells are prepared for pulsed-field gel electrophoresis by lysing cells in situ in an agarose plug.  Following digestion with an appropriate restriction enzyme, the plug is loaded directly into the well of a pulsed-field gel or it can be melted before loading. - [Read Preparation of DNA for Pulsed-field Gel Electrophoresis: Isolation of DNA from Mammalian Cells]

Category: Signalling Signal Transduction Protocols

Protocol provides a method for acheiving a sufficient sample for several determinations of cAMP. The protocol described for measuring the content of cyclic adenosine 3',5'- monophosphate (cyclic AMP or cAMP) in cardiac myocytes is an enzyme-linked immunoassay system. Protocol includes information on: Treatment of Cells and Preparation of Extracts; Use of Environmental Chamber; Reagents and Materials. - [Read Preparation of Myocyte Lysates for Cyclic AMP Determination]

Category: Signalling Signal Transduction Protocols

The protocol provides a method to achieve a sample sufficient for one determination of cAMP using the acetylation protocol. The protocol describes the method used for measuring the content of cyclic adenosine 3',5'- monophosphate (cyclic AMP or cAMP) in RAW 264.7 cells using an enzyme-linked immunoassay system. Information included in the protocol: Treatment of Cells and Preparation of Extracts; Reagents and Materials. - [Read Preparation of RAW 264.7 Lysates for Cyclic AMP Determination]

Category: Fungi Protocols: Neurospora Protocols

Protocol guide for the N. crassa yeast artificial chromosome library. Includes: Chromosome Walking; Hybridization screening of the YAC library; YAC restriction mapping and contig building; Preparation of chromosomal DNA plugs of YAC clones; Partial restriction enzyme digestion of YAC DNA plugs; Using CHEF gel analysis to resolve YAC clones; Southern Hybridization; Isolation of terminal restriction fragments from cloned DNA inserts in YAC clones; etc. - [Read Protocol Guide for the N. crassa Yeast Artificial Chromosome Library]

Category: Molecular Biology Protocols: Restriction Enzyme Digestion

REBASE - The Restriction Enzyme Database - [Read Rebase The Restriction Enzyme Database]

Category: Molecular Biology Protocols: Restriction Enzyme Digestion

Restriction Digestion Methods. Donis-Kellor lab manual. Restriction Enzyme Digests, Restriction Enzyme Buffers, Restriction Digestion of Plasmid, Cosmid, and Phage DNAs. - [Read Restriction Digestion Methods]

Category: Nucleic Acid Electrophoresis Protocols: Pulsed Field Gel Electrophoresis Protocols

Protocol for restriction endonuclease digestion of DNA in agarose plugs. Genomic DNA isolated from mammalian, yeast, or bacterial cells can be digested with restriction endonucleases by incubating agarose plugs containing the DNA in the presence of the desired enzyme.  After digestion, the DNA can be fractionated by pulsed-field gel electrophoresis and either isolated from the gel or analyzed by Southern Hybridization. - [Read Restriction Endonuclease Digestion of DNA in Agarose Plugs Protocol]

Category: Molecular Biology Protocols: Restriction Enzyme Digestion

Digesting DNA with Restriction Endonucleases introduction. Analytical and Preparative reaction mixes. All about restriction enzymes. Pizzorno, BIOL 327. - [Read Restriction Enzyme Digestion of DNA]

Category: Molecular Biology Protocols: Restriction Enzyme Digestion

How much DNA to digest? Master mix for restriction digest. Double digests. Star activity. Matt Lewis, Department of Pathology, Univ. Liverpool. - [Read Restriction enzyme digestion of DNA: basic method]

Category: Molecular Biology Protocols: Restriction Enzyme Digestion

Restriction Enzyme Troubleshooting Guide. Fermentas. Problems: Undigested or incompletely digested DNA, additional bands atypical, Diffused DNA zones, Low fragment ligation efficiency. - [Read Restriction Enzyme Troubleshooting Guide]

Category: Nucleic Acid Detection Protocols

Restriction landmark genomic scanning (RLGS) is a method to detect large numbers of restriction landmarks in a single experiment.  It is based on the concept that restriction enzyme sites can serve as landmarks throughout a genome.  RLGS uses direct end-labeling of the genomic DNA digested with a rare-cutting restriction enzyme and high-resolution two-dimensional electrophoresis. - [Read Restriction Landmark Genomic Scanning Protocol]

Category: DNA Protocols: SAP Protocols

Shrimp Alkaline Phosphatase Protocol. USB. Protocol for Dephosphorylation of 5'-ends of DNA in Restriction Enzyme Reaction. Protocol for Dephosphorylation of 5'-ends of DNA. - [Read Shrimp Alkaline Phosphatase Protocol]

Category: DNA Protocols: SNP Protocols

Compare restriction patterns of two sequences for SNP/Mutation detection. Online program that compares restriction enzyme digest of DNA and finds the differences between two sequences. - [Read SNP Restriction Digestion Comparison]

Category: Reporter Gene Assays: Luciferase Assay Protocols

Protocol describes a split luciferase complementation assay used to study the interaction of proteins in cells. In the split protein strategy, a single reporter protein/enzyme (firefly luciferase [Fluc]) is cleaved into amino-terminal and carboxy-terminal halves; each half is fused to one of two interacting proteins, X & Y. Physical interactions between the two proteins reconstitute the functional reporter protein, leading to enzymatic activities that can be measured by in vitro or in vivo assay - [Read Split Luciferase Complementation Assay for Studying Interaction of Proteins X and Y in Cells]

Category: Pharmacology and Toxicology Protocols: Drug Discovery

Protocol describes a target selective S. aureus whole cell assay that combines agar-diffusion and protein over expression techniques.  This agar based two-plate differential sensitivity assay was used to help confirm the newly discovered antibiotic platensimycin inhibited bacterial growth by specifically targeting the essential FASII enzyme FabF6. - [Read Target Specific Whole Cell Assay for Antibacterial Drug Discovery Protocol]

Category: Genetics and Genomics: Genotyping Protocols: PCR Genotyping

This protocol is designed to detect sequences in the murine genome by polymerase chain reaction amplification, and is adapted from Stratman and Simon (Transgenic Res. 12, 521-522 (2003)).For those familiar with PCR genotyping, this method differs from the typical protocol by utilizing a unique enzyme (Klentaq), 30mer primers, and a 68° annealing temperature. - [Read Universal Mouse Genotyping Protocol]