experiments Protocols

Category: Viral Culture and Isolation Protocols: Viral Isolation Protocols

Protocol describes typical methods that are used to propagate and purify AAV vectors for experiments both in vitro and in vivo. Includes: Principles of the Triple Plasmid Transfection System; Plasmids; Transfection and Extraction of Virus; Purification of the AAV vector. - [Read A Protocol for AAV Vector Production and Purification]

Category: Cloning Protocols: Vector Protocols

Protocol is used to propagate and purify AAV vectors for experiments both in vitro and in vivo. Includes: Principles of the Triple Plasmid Transfection System; Plasmids; Transfection and Extraction of Virus; Purification of the AAV vector. - [Read AAV Vector Production and Purification Protocol]

Category: Plant Biology Protocols: Arabidopsis Tissue Culture

Experiments included for analysis of plant epidermis: Steroid induction of gene expression in Arabidopsis; Replica Molds and Casts of the plant epidermis. - [Read Analysis of the Plant Epidermis Experiments]

Category: Transfection Protocols: Calcium Phosphate Transfection Protocols

This calcium phosphate transfection method works best in cell lines that are 1) highly transformed and 2) adherent (Hela, U2OS, SAOS2, AdAH, NPC-KT and obtain from 20% to 100% transfection efficiency depending on the cell line). Works well for transient experiments but precautions should be used in the design and interpretation of experiments based on the discussion below. Also works very well for generating stable cell lines. This method is quite sensitive to the amount of input plasmid. - [Read Calcium Phosphate Transfection Method]

Category: Epigenetics and Methylation Protocols: ChIP Chromatin Immunoprecipitation Protocols

Chromatin Immunoprecipitation Protocol for Histone Modification Chromatin and Associated Proteins. Roderick O’Sullivan & Joost Martens. Chromatin Immunoprecipitation (ChIP) experiments are routinely performed in many laboratories around the world to examine the occupancy of proteins or chromatin modifications over particular stretches of the genome. - [Read Chromatin Immunoprecipitation Protocol for Histone Modification Chromatin and Associated Proteins]

Category: Immunology: Immunoprecipitation Protocols: Protein Immunoprecipitation Protocols

Protocol describes a denaturing immunoprecipitation (IP) for mammalian cells. Prefer to use denaturing IPs to recover labeled proteins from pulse-chase experiments. The use of denaturing IPs reduces background considerably. - [Read Denaturing Protein Immunoprecipitation from Mammalian Cells Protocol]

Category: siRNA and RNAi Protocols

DSRNAI EXPERIMENTS IN DROSOPHILA S2 CELL CULTURE. using T7 to create RNA. Carolyn Worby. Dixon Lab RNAi experiments. - [Read DSRNAI EXPERIMENTS IN DROSOPHILA S2 CELL CULTURE]

Category: DNA Protocols: DNA Cloning

Preparation of oligo solutions PCR experiments, Digestion of insert DNA, Digestion and dephosphorylation of vector DNA, Ligation of DNA fragments with sticky ends, Ligation of DNA fragments with blunt ends, Preparation of chemically competent E. coli - [Read EMBL Cloning protocols]

Category: Developmental Biology: Utilizing Model Organisms Protocols

In the experiment there are three players A, B, and C. Player A gets an initial endowment of 10, player B gets 0 and player C gets 5 points. First, player A decides how many of his 10 points to transfer to B. Then, player C observes the decisions of A and gets the possibility to leave the payoffs unaffected or to punish A by deducting 3 (or 6) points from A at a cost of 1 point (or 2 points) for C.... - [Read Experiments on Parochial Altruism in Humans; Procedures and Instructions]

Category: DNA Protocols: EMSA DNA-Protein Protocols

This protocol works well for HSF DNA-binding activity experiments. - [Read Gel Mobility Shift Assay in PDF format]

Category: Cytoskeleton Protocols: Actin Myosin Protocols

The same GFP-tagged actin construct used in cell transfection experiments has been used to produce transgenic mice. Transgenic animals allow the imaging of brain tissue in the intact animal, as acutely cut slices or as organotypic slice cultures. They also serve as a source of cells for imaging neurons at high resolution in dispersed low-density cell culture. In contrast to cells transfected in culture, where the level of actin-GFP expression in neurons varies considerably, transgenic mice... - [Read Imaging Actin in Tissue Slices from Transgenic Mouse Brain Protocol]

Category: Western Blot Protocols: General Western Blot Methods Protocols

For immunoblotting experiments, it is often important to compare the total amount of an antigen from many different sources or to learn if a particular source has the antigen under study.  In the approach described here, tissue cultures, bacteria, yeast cells, tissues, and other sources of antigens are disrupted directly in an electrophoresis sample . - [Read Immunoblotting: Preparing Cell Lysates Protocol]

Category: Xenopus Protocols

Explant is very useful for experiments in planar neural induction and convergent extension. - [Read Keller Sandwich Explant 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: DNA Protein Interactions Protocols: Dnase Footprinting Assay Protocols

Quick protocol to give a representative G+A ladder for DnaseI footprint experiments. - [Read Maxam Gilbert Sequencing for DnaseI Footprinting Protocol]

Category: Immunology: Immunoprecipitation Protocols: Protein Immunoprecipitation Protocols

Protocol describes a nondenaturing immunoprecipitation (IP) for mammalian cells. Prefer to use denaturing IPs to recover labeled proteins from pulse-chase experiments. However, the nondenaturing protocol is useful when one wishes to separate soluble from insoluble proteins, or when the antibody being used recognizes a native epitope. - [Read Nondenaturing Protein Immunoprecipitation from Mammalian Cells Protocol]

Category: PCR Protocols

DNA prepared by PCR-mediated gene disruption can be used to transform yeast in gene replacement experiments.  This protocol uses two primers, tailed with approximately 50 nucleotides homologous to the gene of interest, that target insertion of the PCR product to that locus.  Each primer ends with a universal sequence that is designed to amplify various selectable markers from plasmid templates. - [Read PCR-Mediated Gene Disruption: One-Step Method Protocol]

Category: Plant Biology Protocols: Plant Tissue Culture

Dodds, JH and LW Roberts 1995 Experiments in Plant Tissue Culture. ... 19-60 In Plant Cell Culture Protocols, Humana Press, Totowa ... - [Read PLANT CELL, TISSUE AND ORGAN CULTURE - 1998]

Category: siRNA and RNAi Protocols

The protocol listed is Drosophila cells in 6 well plates and our pre-aliquoted 384 well plates. RNAi experiments may be done in other size plates, just scale up or down. Drosophila RNAi Screening Center - [Read RNA Interference Screening (RNAi) in Cultured Cells]

Category: RNAi Technology Protocols: RNAi C. elegans Protocols

Protocol for RNAi screens in C. elegans in a 96-well liquid format and their application to the systematic identification of genetic interactions. The procedure allows thousands of RNAi feeding experiments to be performed per investigator per day. - [Read RNAi Screens in C. elegans Protocol]

Category: Cytogenetics Protocols

Protocol describes methods for maintaining healthy, dividing mammalian cells in culture and during imaging, when mitosis can be examined. Rose chambers are preferable for observation and microinjection of living mitotic cells, but slide/coverslip preparations are easy to make and do not require any special equipment. Another inexpensive and easy-to-use alternative is to grow cells in a culture dish with a glass bottom. Such dishes are suitable for microinjection experiments. - [Read Studying Mitosis in Cultured Mammalian Cells Protocol]

Category: Cell Biology and Cell Culture: Cell Cycle Protocols: Mitosis Protocols

Protocol describes methods for maintaining healthy, dividing mammalian cells in culture and during imaging, when mitosis can be examined. Rose chambers are preferable for observation and microinjection of living mitotic cells, but slide/coverslip preparations are easy to make and do not require any special equipment. Another inexpensive and easy-to-use alternative is to grow cells in a culture dish with a glass bottom. Such dishes are suitable for microinjection experiments. - [Read Studying Mitosis in Cultured Mammalian Cells Prtocol]

Category: Yeast Cell Culture Protocols: Yeast Sporulation Protocols

This protocol describes dissection of yeast tetrads. Tetrad analysis is useful for linkage studies and for constructing strains for genetic and biochemical experiments. - [Read Tetrad Dissection Protocol]

Category: Reporter Gene Assays: Luciferase Assay Protocols

Transfection of primary leukocytes has traditionally been a challenging but much desired protocol. It allows not only the analysis of cells in a more natural state to a cell line system, it enables the direct comparison of, for e.g. transcriptional activity using luciferase reporters, in immune cells taken from genetically-altered mice. In addition, importantly it allows for "rescue experiments" in knockout cells & the ability to over-express or reconstitute wild-type and/or mutated constructs. - [Read Transfection of Bone Marrow-Derived Mast Cells for Transcription Factor Luciferase Reporter Assays]

Category: Immunohistochemistry Protocols: Antigen Retrieval Protocols

Fixation can mask epitopes. However, it is often possible to re-expose them using a gentle incubation with proteases, which removes obstructing structures and allows antibody access, as described here. Many proteases can be used for this procedure, including very crude preparations of proteases, such as pronase. However, using a better-characterized protease, such as trypsin, allows a more controlled reaction and better comparison between experiments. - [Read Unmasking Hidden Epitopes with Proteases Protocol]