methods Protocols

Category: Microinjection Protocols: Microinjection Equipment

This protocol contains methods for pulling microinjection needles using two different models of pipette pullers. The advantage of pulling needles in the laboratory is that a variety of different needle types can be pulled, depending on the samples and cells being injected. An added advantage is cost; once a pipette puller has been purchased, boxes of glass capillaries are inexpensive compared to premade microinjection needles. - [Read Preparation (Pulling) of Needles for Gene Delivery by Microinjection Protocol]

Category: Microinjection Protocols: Microinjection of Proteins Protocols

his protocol provides methods for the preparation of protein samples and for loading them into pulled microinjection pipettes. Stock solutions of proteins are thawed, diluted (if desired), centrifuged at high speed to remove aggregates, and kept on ice until loading. Loading into micropipettes can be done using either a "front-loading" or a "backfilling" procedure. - [Read Preparation and Loading of Protein Samples for Microinjection Protocol]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols

A procedure for direct and indirect staining of single-cell suspensions of lymphoid tissue or peripheral blood lymphocytes to detect cell surface membrane antigens is presented. In addition, support protocols present methods for fluorescence labeling of purified antibodies. A protocol for flow cytometric analysis of intracellular antigens in single-cell suspensions is also included. - [Read Preparation of Cells and Reagents for Flow Cytometry Protocols]

Category: Microbiology: Bacterial Competent Cells Preparation E.Coli

Simple E.coli competent cell Protocol. Based on a protocol from Neil Olszewski and Scott Medberry.. Craig D. Amundsen - [Read Preparation of Competent Bacterial Cells]

Category: Cell Organelle Protocols: Subcellular Fractionation Protocols

The employment of differential centrifugation to prepare crude fractions of subcellular particles from homogenates is often a necessary first step to a subsequent purification of one or more particles on a density gradient. This protocol describes the use of differential centrifugation to fractionate a mammalian liver homogenate but similar methods should be applicable to all mammalian tissues and cultured cells. - [Read Preparation of Crude Subcellular Fractions by Differential Centrifugation Protocol]

Category: Viral Manipulation Protocols: M13 Phage Manipulation Protocols

The double-stranded replicative form (RF) of bacteriophage M13 is isolated from infected cells using methods similar to those used to purify plasmid DNA. Several micrograms of RF DNA can be isolated from a 1-2-ml culture of infected cells. - [Read Preparation of Double-stranded (Replicative Form) Bacteriophage M13 DNA Protocol]

Category: Stem Cell Protocols: Stem Cell Injection Protocols

Injection of ES cells into the cavity of blastocysts is one of the main methods for generating chimeras. This protocol describes the preparation of ES cells for injection. - [Read Preparation of Embryonic Stem (ES) Cells for Injection Protocol]

Category: Cell Biology and Cell Culture: Endothelial Cell Protocols

Endothelial cells, which line blood vessels, can be prepared from a variety of tissues. They are frequently prepared from the umbilical vein, which is relatively easy to obtain. The procedure is clearly described and provides a large population of highly purified endothelial cells. There are also methods for obtaining endothelial cells from other tissues such as fat, skin, and mucosa. These methods require special care and generate smaller populations of cells. - [Read Preparation of Endothelial Cells Protocol]

Category: Cytogenetics Protocols: Fluorescent In Situ Hybridization FISH Protocol

Preparation of metaphase chromosomes from lymphoblastoid cell lines for FISH analysis. Sanger Institue. - [Read Preparation of metaphase chromosomes from lymphoblastoid cell lines]

Category: Cytogenetics Protocols

Protocol for the preparation of metaphase spread. Includes three methods. - [Read Preparation of Metaphase Spread Protocol]

Category: Radioactivity: Liquid Scintillation Counting

Excellent guide for Liquid Scintillation Counting. Includes protocols and methods for counting gel slices, SPECIAL SAMPLE PREPARATION PROTOCOLS: TLC Plates, protocol for Counting Samples on Cellulose-ester, Filters (MilliporeTM filters), Counting Tissue radioacitivity, Counting 14CO2, Samples in Polyacrylamide Gels. National Diagnostics. National Diagnostics Laboratory Staff. - [Read Principles and Applications of Liquid Scintillation Counting PDF]

Category: Viral Manipulation Protocols: Phage Protocols

Protocol describes methods to superinfect bacteria carrying a recombinant phagemid with a high-titer stock of an appropriate helper virus and to assay the yield of filamentous virus particles that carry single-stranded copies of the phagemid DNA. The key to success in using phagemids is to prepare a stock of helper virus whose titer is accurately known. - [Read Producing Single-stranded DNA with Phagemid Vectors Protocol]

Category: Immunology: T Cell Protocols

Provides methods for the derivation of specific types of T cell clones: preparation and maintenance of alloreactive murine helper T (TH) lymphocyte and cytotoxic T lymphocyte (CTL) clones using the limiting dilution technique and derivation of TH clones reactive with soluble protein antigens including a method for the selection of either TH1 or TH2 lymphocyte subsets. - [Read Production of T Cell Clones Protocol]

Category: Protein Protocols: Protein Precipitation Procols

Laboratory sample cleanup is a necessary part for analytical preparation analysis. The removal of Contaminants such as proteins, cell debris and other materials is an important step. Typically this has been done by using Acetonitrile and then Centrifugation to pellet the debris leaving the clean supernant. After this process supernatant can be used for further analysis by HPLC, GC, MS and other analysis tandem methods. HTS Labs. - [Read Protein Precipitation Microplate]

Category: Protein Protocols: Protein Trafficking

Methods on protein trafficking. Information on N5 Secretion and protein trafficking. Methods include: Alcian Blue Test; Lucifer Yellow uptake assay; Preparation of total protein extracts for western immunoblots; Screen for Drug Sensitivity. - [Read Protein Trafficking Methods]

Category: Mass Spectrometry Protocols

Colloidal coomassie blue staining, Destaining, Reduction/Alkylation, Peptide extraction For MALDI-MS analysis and For MALDI-MS analysis protocols and methods. Institut Curie Paris. - [Read Proteomic protocols and Peptide Extraction]

Category: Cell Biology and Cell Culture

Microtubule binding assay protocol using the materials: Siliconized ultracentrifuge microfuge tubes, GTP-depleted microtubules, 6X SDS loading dye, 1X SDS loading dye, Coomassie Brilliant Blue R 250 (0.8% in 50% methanol + 10% acetic acid)and Destaining solution (13% methanol + 3% acetic acid). - [Read Protocol for Microtubule Binding Assays]

Category: Cell Biology and Cell Culture

This protocol introduces the use of a liquid-filled wash chamber that separates unbound cells by gravity thereby eliminating uncontrolled shear forces and passage of adherent cells through a liquid/air interface. The cells are loaded with a fluorescent dye (6-carboxyfluorescein diacetate) for detection although other methods such as radioactive labels malabels may be used. This protocol is also useful for assaying molecules that promote or inhibit cell adhesion. - [Read Protocol for Measurement of Cell Adhesion Under Static Conditions]

Category: Cell Biology and Cell Culture

Protocol for motility using VE-DIC microscopy. Materials required: Anti-GST (glutathione S-transferase) antibody, PB buffer =10 mM NaPO4 pH 7.2, EGTA, MgCl2, Clarified motor lysate, MTs or Axoneme-MTs,Mg·ATP, VALAP (1 Vasoline: 1 Lanolin: 1 Paraffin, heated gently until melted) and Maxell XR-S120 Black Magnetite Super-VHS tapes or comparable. - [Read Protocol for Motility using VE-DIC Microscopy]

Category: Cell Biology and Cell Culture

Protocol for motor polarity using fluorescent microtubules using the materials: Rhodamine-tubulin (Molecular Probes),NEM- tubulin, PC-tubulin, Mg·GTP, MgCl2 and Glycerol. - [Read Protocol for Motor Polarity Using Fluorescent Microtubules]

Category: Protein Protocols: Protein Extraction Protocols

Protocol for Protein Extraction Using Proteomics. Extraction of proteins from plant cells that are rich in compounds that interfere with the 2-Dimensional electrophoretic separation methods such as salts, organic acids, phenolics, pigments, terpenes, among others. A common protocol used in our lab for extraction proteins from plant tissues consists in the homogenization of mortar-grounded material in liquid nitrogen with an extraction buffer. - [Read Protocol for Protein Extraction Using Proteomics]

Category: Cell Biology and Cell Culture

Protocol for Standard Fluorescence Motility Assay. Materials requires: Microtubules, Flow Cell, Standard Solutions: BRB80, BRB80T, BRB80CS0.5, BRB80CA, BRB80CT - [Read Protocol for Standard Fluorescence Motility Assay]

Category: Cytoskeleton Protocols: Intermediate Filaments Protocols

Intermediate filaments (IF) are major cytoskeletal systems of vertebrate and many nonvertebrate cells whose expression is cell-type specific and developmentally regulated. This protocol describes a method for purifying one type of IF, vimentin, from bovine lens tissue. Purification of human vimentin expressed in Escherichia coli is also described. These methods are useful in the preparation of other IF protein subunits for microinjection studies as well. - [Read Purification of Bovine Lens and Bacterially Expressed Human Vimentin Protocol]

Category: Cytogenetics Protocols: Array CGH

Random Labelling DNA for array CGH Protocol. Sanger Institute - [Read Random Labelling DNA for array CGH Protocol]

Category: DNA Protocols: DNA Extraction Protocols: Agarose Gel DNA Extraction Protocols

Rapid Elution of DNA Agarose Gels Protocol.  This method allows quick purification of DNA fragments from agarose gels for use in cloning and other reactions. Higher yields of purified DNA can be obtained from commercially available purification kits, however greater ligation efficiencies per given amount of DNA have been seen with the use of these described spin columns. Hahn Lab. - [Read Rapid Elution of DNA Agarose Gels Protocol]