methods Protocols

Category: PCR Protocols: Quantitative PCR Protocols

General guidelines for long-PCR conditions and enzyme mixtures. Efficient long-PCR results from the use of two polymerases: a non-proofreading polymerase is the main polymerase in the reaction, and a proofreading polymerase (3' to 5' exo) is present at a lower concentration. Includes: For PCR with low-complexity templates (e.g., plasmid and cosmid inserts); For PCR with moderate-complexity templates (e.g., bacterial genomic DNA); For PCR with high-complexity templates (e.g., human genomic DNA). - [Read Long-PCR Reagents and Guidelines]

Category: Plant Biology Protocols

Protocol for the lyophilization of cotton leaves. - [Read Lyophilization of Cotton Leaves]

Category: Protein Microarray Protocols

Lysate Microarrays. High throughput methods that detect abundance and post-translational modification. MacBeath Lab - [Read Lysate Microarrays - Information]

Category: Bacterial Cell Culture Protocols: Bacterial Stocks Protocols

Plasmid (pUC series) containing genomic DNA fragments are maintained in E. coli strain DH5aTM. The E. coli cultures are routinely cultured at 37 C on Luria-Bertani (LB) agar on or in LB broth containing Ampicillin (30 µg/ml) or Carbenicillin (50 µg/ml broth, 100 µg/ml agar). E. coli strains are usually preserved in stab agar or glycerol for mid-term storage and lyophilized for long-term storage. - [Read Maintenance of Probes in Bacteria Including Escherichia coli Protocol]

Category: Xenopus Protocols: Xenopus Injection Protocols

Protocol for making and injecting Xenopus embryos. Includes: Injections; Egg Activation and Inversion Experiments; - [Read Making and Injecting Xenopus Embryos Protocol]

Category: Primary Cell Isolation and Culture Protocols

There are several manual methods that can be used to perform tissue microdissection. Techniques using hand-held tools as well as mechanical micromanipulator-based approaches have been described. However, speed and precision are the most important parameters and any method that achieves these is adequate. Investigators should also expect to invest time initially by practicing on 10 to 20 cases to begin to feel comfortable with the technique. - [Read Manual Microdissection]

Category: Signalling Signal Transduction Protocols: MAPK Protocols

MAPK immunoprecipitation protocol and methods. Upstate. - [Read MAPK immunoprecipitation Protocol PDF]

Category: Cell Biology and Cell Culture: Apoptosis Protocols: Apoptosis Analysis

Measurement of Apoptosis and Other Forms of Cell Death. Jagan Muppidi, Melissa Porter, and Richard M. Siegel. As programmed cell death (PCD) or apoptosis has emerged as an important regulator of development and homeostasis in multicellular organisms, methods to quantify apoptosis and to distinguish it from necrosis have been developed. This unit presents a set of assays for these purposes, many of which are technically very simple and ideally suited to the study of hematopoietic cells. - [Read Measurement of Apoptosis and Other Forms of Cell Death]

Category: Signalling Signal Transduction Protocols: Calcium Measurement Protocols

Phenomenal review of the methods and references to intracellular calcium measurements. TAKAHASHI et al Phys. Rev. 1999 - [Read Measurement of Intracellular Calcium PDF]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: Intracellular Ion Measurement Protocols

Describes flow cytometric protocols using the dyes Indo-1 AM, Fluo-3, and Fura Red AM to measure intracellular calcium concentration. Support protocols detail the use of calcium buffers to calibrate a flow cytometric calcium assay, and methods to facilitate dye loading; an alternate protocol describes the use of a spectrofluorimeter to measure intracellular calcium for those investigators without access to a flow cytometer. - [Read Measurement of Intracellular Ions by Flow Cytometry Protocol]

Category: Yeast Cell Culture Protocols: Yeast Media, Solutions and Stocks Protocols

The yeast, Saccharyomyces cerevisiae, has become an important organism in molecular, biochemical, and genetic analysis. The organism has specific requirements for growth under a variety of conditions. The media, both liquid and solid, simple, define, and complex are describe in this unit. Also included are methods for handling, storing, and shipping stock of yeast. - [Read Media and Culture of Yeast Protocol]

Category: Cell Biology and Cell Culture: Cell Culture Growth Media: Yeast Cell Culture Growth Media Protocols

Protocol for media for culture of Aspergillus nidulans. Includes minimal and complete media. - [Read Media for Culture of Aspergillus nidulans Protocol]

Category: Fungi Protocols: Aspergillus Protocols

Protocol for media for culture of Aspergillus nidulans. Includes: Minimal medium; Complete media. - [Read Media for Culture of Aspergillus nidulans Protocol]

Category: Cell Biology and Cell Culture: ES Stem Cell Biology and Culture: MEF Protocols Mouse Embryonic Fibroblasts

MEFs. Thompson Lab Stem Cell Methods and Procedure. Irradiation and plating MEFs, Splitting MEF cells, Thawing MEF, Freezing and Harvestion MEF cells. - [Read MEF Mouse Embryonic Fibroblast Protocols]

Category: Nucleic Acid Modification Protocols: DNA Methylation Protocols

Methods for analyzing the role of DNA methylation and chromatin structure in regulating T lymphocyte gene expression. - [Read Methods for Analyzing the Role of DNA Methylation and Chromatin Structure in Regulating T Lymphocyte]

Category: DNA Protocols: DNA Extraction Protocols

Large scale double-stranded DNA isolation, Miniprep double-stranded DNA isolation, Large scale M13RF isolation, Single-stranded M13 DNA isolation using phenol. Minion Lab.Veterinary Medicine Iowa Univ. - [Read Methods for DNA isolation]

Category: DNA Protocols: DNA Extraction Protocols

Large scale double-stranded DNA isolation, Midiprep double-stranded DNA isolation. Bruce A. Roe, Department of Chemistry and Biochemistry, The University of Oklahoma, Norman. - [Read Methods for DNA isolation - Protocol Book]

Category: Cell Culture Protocols: Insect Cell Culture Protocols

Insect cell cultures are now commonly used in insect physiology, developmental biology, pathology, and molecular biology. As the field has advanced from methods development to a standard procedure, so has the diversity of scientists using the technique. This paper describes methods that are effective for maintaining various insect cell lines. The procedures are differentiated between loosely or non-attached cell strains, attached cell strains, and strongly adherent cell strains. - [Read Methods for Maintaining Insect Cell Cultures]

Category: Plant Biology Protocols: Plant DNA RNA Isolation Protocols

AMES/Chloroform extraction to extract total RNA from potato leaves for viroid analysis. - [Read Methods for Plant RNA Isolation]

Category: Neuroscience Protocols

Methods for structure function studies of neurotrophic factors. - [Read Methods for Structure Function Studies of Neurotrophic Factors]

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

Exponentially growing cells are asynchronous with respect to the cell cycle stage. Detection of cell cycle-related events is improved by enriching the culture for cells at the stage during which the particular event occurs. Methods for synchronizing cells are provided here, including those based on morphological features of the cell. - [Read Methods for Synchronizing Cells at Specific Stages of the Cell Cycle]

Category: Cell Biology and Cell Culture

Protocol on microtubule assembly using purified tubulin. - [Read Microtubule Assembly Protocol]

Category: Molecular Imaging: Immunofluorescence Microscopy

Minimizing Background in Immunofluorescence Procedures. Includes tips and methods on reducing background staining. Janis V. Giorgi Flow Cytometry Laboratory, UCLA. - [Read Minimizing Background in Immunofluorescence Procedures]

Category: Plant Biology Protocols: Plant DNA RNA Isolation Protocols

Protocol for the isolation of mitochondrial DNA. - [Read Mitochondrial DNA Isolation Protocol]

Category: Plant Biology Protocols

The standard protocol for in situ hybridizations in plants still involves fixing fresh tissue, embedding the tissue in wax, sectioning with a microtome and detection of the transcripts of interest using labeled RNA-probes. This protocol concentrates only on nonradioactive methods, as they are easy to perform, very sensitive and even faster than techniques involving radioisotope labels. - [Read Molecular and Biochemical Analysis of Arabidopsis Protocol]