conditions Protocols

Category: PCR Protocols: In-situ PCR Protocols

96-well RNA In Situ Hybridization Protocol. Probe Preparation, Cell Inoculation, PCR, RNA Probe Preparation, etc, Very In-depth Protocol and Method Conditions. Berkeley Drosophila Genome Project. - [Read 96-well RNA In Situ Hybridization Protocol]

Category: Microscopy Protocols: Live-Cell Imaging Protocols

This protocol describes a sealed preparation that allows the continuous long-term observation of cultured mammalian cells on upright or inverted microscopes without environmental CO2 control. The preparation allows for optical conditions consistent with high-quality imaging and good cell viability for at least 100 hours. - [Read A Sealed Preparation for Long-Term Observations of Cultured Cells]

Category: Primary Cell Isolation and Culture Protocols: Mammalian Cell Culture Protocols

There are many ways to adapt cell lines to serum-free media. Five methods are presented that are designed for adapting hybridomas to a protein-free medium. These protocols may require some modifications for your particular cell line and conditions. - [Read Adapting Cells to a Serum-Free Environment Protocol]

Category: Microbiology: Antibiotics Concentration in Media LB

Antibiotic stock solutions preparation, Storage conditions for antibiotics, Working Concentrations for high copy plasmids. Kay Schneitz Univ Zurich. - [Read Antibiotics]

Category: Cell Culture Protocols: Sterile Technique Protocols

This unit describes some of the ways that a laboratory can deal with the constant threat of microbial contamination in cell cultures. A protocol on aseptic technique is described first. This catch-all term universally appears in any set of instructions pertaining to procedures in which noncontaminating conditions must be maintained. - [Read Aseptic Technique for Cell Culture Protocol]

Category: Reporter Gene Assays: B-Galactosidase Assay Protocols

Method describes how a crude extract is prepared, and the activity is normalized to the amount of protein assayed. This method is particularly suitable for comparing cells that are grown under very different conditions or that have different genetic backgrounds. - [Read Assay of ß-Galactosidase in Yeast: Assay of Crude Extracts]

Category: Yeast Protocols: Yeast Genetics Protocols: B-Galactosidase Assay Protocols

Protocol describes how a crude extract is prepared, and the activity is normalized to the amount of protein assayed. This method is particularly suitable for comparing cells that are grown under very different conditions or that have different genetic backgrounds. - [Read Assay of ß-Galactosidase in Yeast: Assay of Crude Extracts Protocol]

Category: PCR Protocols: Colony PCR

PCR Reaction Conditions for Colony PCR. Contributed by Lynn Hancock. Enterococcus Research Site, The Board of Regents of the University of Oklahoma. - [Read Colony PCR Protocol]

Category: Pharmacology and Toxicology Protocols: Cytotoxicity Protocols: Cytotoxicity Assays in Cell Culture Protocols

The potential cytotoxicity of compounds under hypoxic conditions is determined by exposing cell cultures to test compounds in a low oxygen atmosphere.  Subsequent cell survival is determined by the MTT and methylene blue colorimetric assays. - [Read Colorimetric Cytotoxcity Assays for Anchorage Dependent Cells Protocol]

Category: Xenopus Protocols: Xenopus Egg Extracts Protocols

Protocol for crude depletion conditions for XKCM1. - [Read Crude Depletion Conditions for XKCM1 Protocol]

Category: Cell Culture Protocols: Cell Storing and Cell Freezing Protocols

Cryogenic preservation (storage below -100°C) of cell cultures is widely used to maintain backups or reserves of cells without the associated effort and expense of feeding and caring for them. The success of the freezing process depends on four critical areas: Proper handling and gentle harvesting of the cultures; Correct use of the cryoprotective agent; A controlled rate of freezing; Storage under proper cryogenic conditions. - [Read Cryogenic Preservation and Storage of Animal Cells Protocol]

Category: Primary Cell Isolation and Culture Protocols: Mammalian Cell Culture Protocols

Culture conditions for various stable cell lines. - [Read Culture Conditions for Various Stable Cell Lines]

Category: Stem Cell Protocols: Stem Cell Culture Protocols

Culture conditions have been established for a second blastocyst-derived cell line, trophoblast stem (TS) cells, in addition to embryonic stem (ES) cells. This protocol describes a method for culturing TS cell lines. These cells can then be used to study trophoblast differentiation and placental function. - [Read Culturing Trophoblast Stem (TS) Cell Lines Protocol]

Category: Pharmacology and Toxicology Protocols: Enzyme Assays Protocols

Protocol for Cytochrome P450. Includes: Microsome Assays General; General Reaction Conditions; Specific Substrates for Cytochrome P450 Isozymes. - [Read Cytochrome P450 Recombinant Enzyme Microsome Protocol]

Category: Transfection Protocols: Stable Transfection Protocols

Pulsed electrical fields can be used to introduce DNA into a wide variety of animal cells. Electroporation works well with cell lines that are refractive to other techniques, such as calcium phosphate-DNA coprecipitation. But, as with other transfection methods, the optimal conditions for electroporating DNA into untested cell lines must be determined experimentally. - [Read DNA Transfection by Electroporation]

Category: Transfection Protocols

Protocol should be viewed as a starting point for systematic optimization of transfection mediated by lipofecting agents. Once a positive signal has been obtained from a transfected plasmid carrying a standard reporter gene, optimal conditions for transfection can be established by systematic variation of parameters such as the initial cell density, the amount and purity of DNA, the media and serum, and the time of exposure of the cells to the cationic-lipid-DNA complex. - [Read DNA Transfection Mediated by Lipofection Protocol]

Category: Drosophila Protocols: Drosophila Culture Media Protocols

Protocol for Drosophila growth conditions. Includes: MAINTENANCE; Semi-adherent cell lines; Adherent cell lines; SL2, S2C, S2*; DL2, DL1, S2R+, Kc167, Clone 8, S3; BG2. - [Read Drosophila Growth Conditions 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: Mouse Protocols: Mouse Genetics Protocols

Protocol describes a method for electroporating DNA into ES cells, as well as selection methods. Pilot studies should be performed to optimize the conditions for each DNA construct. The selection method described here is one of the most complex. It involves targeting constructs in which the bacterial neomycin-resistance gene disrupts the coding sequence of the mouse gene. - [Read Electroporating DNA into Embryonic Stem (ES) Cells and Selection Methods Protocol]

Category: PCR Protocols: PCR Optimization

Method for amplifying DNA enzymatically by the polymerase chain reaction (PCR), including procedures to quickly determine conditions for successful amplification of the sequence and primer sets of interest, and to optimize for specificity, sensitivity, and yield.  The first step of PCR simply entails mixing template DNA, two appropriate oligonucleotide primers, Taq or other thermostable DNA polymerases, deoxyribonucleoside triphosphates (dNTPs), and a buffer. - [Read Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization Protocol]

Category: Bacterial Cell Culture Protocols

The growth conditions of microbial cell cultures and the time of sample collection should be optimized and standardized when growing cells for protein extraction. Because cells may excrete proteases and other extracellular enzymes, and compounds in the medium may interfere with extraction, wash cultures with an isotonic buffer, such as PBS or sucrose before solubilization. - [Read Extraction and Solubilization of Total Protein from Microorganisms Protocol]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols

Flow cytometric analysis of gram-negative bacterial cells. Includes: Staining Conditions and FACSort Settings; Light Scatter Resolution on the BD FACSort; Bacterial Cell Resolution on the BD FACSort. - [Read Flow Cytometric Analysis of Gram-Negative Bacterial Cells]

Category: Cell Organelle Protocols: Subcellular Fractionation Protocols: Membrane Isolation Protocols

The protocol described in this protocol has been used principally for analyzing the Golgi, endoplasmic reticulum and trans-Golgi network but markers for other compartments (e.g. ERGIC and endosomes) have also been analyzed. Modifications either to the gradient density range or the centrifugation conditions influence the ability of the gradient to resolve multiple compartments. - [Read Fractionation of Golgi, ER, TGN and Other Membrane Compartments in Pre-Formed Iodixanol Gradients]

Category: DNA Protein Interactions Protocols: Electrophoretic Mobility Shift Assays EMSA Protocols

Protocol for gel mobility shift assay conditions -Mg/EDTA in gel and buffer. - [Read Gel Mobility Shift Assay Conditions -Mg/EDTA in Gel and Buffer Protocol]

Category: RNA Protocols: RT-PCR and cDNA synthesis

Gene-specific RT-PCR. Used for microdissected RNA, but may apply to other conditions. Molecular Profiling Initiative, NCI. - [Read Gene-specific RT-PCR]