important Protocols

Category: Plant Biology Protocols: Plant Proteins, Peptides and Antigens

The pH is an important parameter controlling many metabolic and signalling pathways in living cells. Recombinant fluorescent pH indicators (pHluorins) have come into vogue for monitoring cellular pH. They are derived from the most popular Aequorea victoria GFP (Av-GFP). Here, we present a novel fluorescent pH reporter protein from the orange seapen Ptilosarcus gurneyi (Pt-GFP) and compare its properties with pHluorins for expression and use in plants. - [Read A Novel Fluorescent pH Probe for Expression in Plants]

Category: Nucleic Acid Purification Protocols

Protocol for a single-step method for the simultaneous preparation of DNA, RNA, and protein from cells and tissues. The yield of total RNA depends on the tissue or cell source, but it is generally in the range of 4-7 µg/mg starting tissue or 5-10 µg/106 cells.  IMPORTANT: Prepare all reagents used in this protocol with Diethyl pyrocarbonate (DEPC)-treated H2O. - [Read A Single-step Method for the Simultaneous Preparation of DNA, RNA, and Protein from Cells and Tissue]

Category: PCR Protocols: Bisulfite Conversion Based PCR Protocols

Protocol for bisulfite-PCR for restriction analysis and/or sequencing. Bisulfite-PCR followed by restriction is a rapid and semi-quantitative method of analyzing DNA methylation.  The PCR products are also suitable for either direct sequencing or cloning and sequencing.  The most important step here is primer selection. - [Read Bisulfite-PCR for Restriction Analysis and/or Sequencing Protocol]

Category: Cell Immortalization Protocols

Information why cell immortalization is important. Also the available tools to be able to perform cell immortalization. - [Read Cell Immortalization]

Category: Protein Protocols: Protein Extraction From Cells

Cell Lysate Extracts. Great protocols for cell lysis preparation from a variety of cell types. There are numerous methods of cell stimulation and lysis. For a given protein, Upstate’s Laboratories determine the specific treatment upon initial testing of its products. It is important to select the correct cell line, stimulation procedure (if any), and lysis protocol. Upstate. - [Read Cell Lysate Extracts]

Category: Molecular Biology Protocols: Restriction Enzyme Digestion

Cloning Enzymes a Guide Promega. An Enzyme Resource Guide series, highlights those enzymes important in nucleic acid cloning procedures. Promega - [Read Cloning Enzymes a Guide Promega]

Category: Cell Biology and Cell Culture: Cell Viability Protocols

Cell-based assays are important tools for contemporary biology and drug discovery because of their predictive potential for in vivo applications.This assay gives ratiometric, inversely proportional values of viability and cytotoxicity (Figure 4.15) that are useful for normalizing data to cell number. Also, this reagent is compatible with additional fluorescent and luminescent chemistries. - [Read Determining Number of Live and Dead Cells in Cell Population: Cytotoxicity Assay Protocol]

Category: BACs Bacterial Artificial Chromosome Protocols

Sequencing conditions tested for the ABI Big-Dye terminators (PE-ABI #4303150 for the 1000 reaction kit - Description: TF,KIT BTD RR-1000) with various templates. Important to quantitate all templates by agarose gel electrophoresis vs size and concentration standards and do a few tests with different template concentrations to determine the optimal conditions for your reactions. Several conditions are given. - [Read Dye Protocols and Notes for Cosmid, BAC, BAC, Fosmid Templates]

Category: Cell Organelle Protocols: Subcellular Fractionation Protocols

Acidocalcisomes, the dense acidic calcium-storing organelles, which were originally identified in Trypanosoma cruzi, have no parallels in mammalian cells. They thus represent a unique functional characteristic, not shared by the host and hence offer an important potential target for chemotherapy of Chagas disease. - [Read Fractionation of Acidocalcisomes and Other Organelles from Trypanosoma, Leishmania, Chlamydomonas]

Category: Stem Cell Protocols

This protocol describes a method for freezing and thawing ES cells using cryovials. It is important to freeze ES cell stocks as soon as possible to reduce the time that they are in culture. A careful record should be kept of the number of times cells are passaged and the location of the cryovials. - [Read Freezing and Thawing of Embryonic Stem (ES) Cells Using Cryovials 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: Cytogenetics Protocols

Karyotyping is a valuable research tool used to determine the chromosome complement within cultured cells. It is important to keep in mind that karyotypes evolve with continued culture. Because of this evolution, it is important for the interpretation of biochemical or other data, that the karyotype of a specific sub-line be determined. - [Read Karyotyping 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: 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: 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: Antibody Protocols: Antibody Production Protocols

Protocol for monoclonal antibody production fusion. Fusion is an important procedure to produce monoclonal antibodies (MoAb). - [Read Monoclonal Antibody Production Fusion Protocol]

Category: Cell Biology and Cell Culture: ES Stem Cell Biology and Culture

Boris Greber. Important rules for ES cell culture, Signs of differentiation, General tips, ES medium, Thawing ES cells, Freezing, Passaging. - [Read Mouse embryonic stem (ES) cell culture - basic procedures]

Category: Cell Culture Protocols: Cell Culture Contamination Prevention and Removal Protocols

One of the most important, but frequently overlooked, cell culture procedures is testing cultures for microbial contamination, especially mycoplasma. It is critical for every cell culture laboratory to only use cell lines that have been carefully screened for mycoplasma. Fortunately, there is a simple fluorochrome DNA staining test that can detect both mycoplasma and virtually any other prokaryote contaminants. - [Read Mycoplasma Detection Using DNA Staining Protocol]

Category: Cell Culture Protocols: Cell Culture Contamination Prevention and Removal Protocols

For both biological and economical reasons, it is important to eliminate mycoplasmas from cell cultures being used for basic research, diagnosis, and biotechnological production. The most commonly used method for elimination, inactivation, or suppression of mycoplasmas in cell cultures is treatment with antibiotics. In general, antibiotic therapies do not result in long-lasting, successful elimination. Also, the cytotoxic properties of antibiotics can cause undesirable side effects on cells. - [Read Mycoplasma Elimination Reagent Protocol]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols

Paraformaldehyde Fixation of Cells protocol. This fixation method is good for cells labeled by fluorochrome-conjugated antibodies to membrane antigens.  It will stabilize the light scatter and labeling for up to a week in most instances, allowing you to be more flexible in scheduling cytometer time.  Furthermore, it inactivates most biohazardous agents, so it is important from a safety standpoint as well. Iowa University. - [Read Paraformaldehyde Fixation of Cells]

Category: E Coli Protocols: E coli Transformation Protocols

Procedure generates competent cultures of E. coli that can be transformed at high frequencies (5 x 108 transformed colonies/µg of superhelical plasmid DNA). IMPORTANT All steps in this protocol should be carried out aseptically. - [Read Preparation and Transformation of Competent E. coli 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: Cell Biology and Cell Culture: Endothelial Cell Protocols

Primary mammalian endothelial cells protocol. This protocol is designed for primary endothelial cells isolated from various organs of mammals. Large and flat cells, often with large nuclei. Includes: Required reagents; DNA preparation and quality; Preparation of cells and cell culture; Important controls; Nucleofection protocol. - [Read Protocol Primary Mammalian Endothelial Cells]

Category: Nucleic Acid Purification Protocols: RNA Isolation Protocols: RNA Isolation fromTissues or Cultured Cells Protocols

Single-step technique, cells are homogenized in guanidnium thiocyanate and the RNA is purified from the lysate by extraction with phenol:chloroform at reduced pH.  Many samples can be processed simultaneously and speedily.  The yield of total RNA depends on the tissue or cell source and is generally in the range of 4-7 µg/ml starting tissue or 5-10 µg/106 cells.  IMPORTANT: Prepare all reagents used in this protocol with Diethyl pyrocarbonate (DEPC)-treated H2O. - [Read Purification of RNA from Cells and Tissues by Acid Phenol-Guanidinium Thiocyanate-Chloroform Extract]

Category: Cell Culture Protocols: Cell Culture Contamination Prevention and Removal Protocols

Quality is important in all aspects of tissue culture since the quality of materials used i.e. media and other reagents) will affect the quality of the cultures and products derived from them. The main areas of quality control that are of concern for tissue culture are: The quality of the reagents and materials; The provenance and integrity of the cell lines; The avoidance of microbial contamination. - [Read Quality Control Considerations for Cell Culture]