experiment Protocols

Category: PCR Protocols: cDNA Synthesis Protocols

An oligodeoxynucleotide primer hybridized to mRNA is extended by an RNA-dependent DNA polymerase to create a cDNA copy that can be amplified by PCR.  Depending on the purpose of the experiment, the primer for first-strand cDNA synthesis can be specifically designed to hybridize to a particular target gene, or a general primer such as oligo(dT) can be used to prime cDNA synthesis from essentially all mammalian mRNAs - [Read Amplification of cDNA Generated by Reverse Transcription of mRNA Protocol]

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

An Integrative Procedure for Apoptosis Identification and Measurement. Yingyu Cui Lab/Group: National Key Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences. I have uploaded an integrative procedure through which a relatively satisfactory result can be obtained following a single stage of cell culture and transient cell treatment, then detection with different instruments. This shortens experiment time. - [Read An Integrative Procedure For Apoptosis Identification And Measurement]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: Apoptosis Cytometry Protocols

An integrative procedure through which a relatively satisfactory result can be obtained following a single stage of cell culture and transient cell treatment, then detection with different instruments. This shortens experiment time. - [Read Apoptosis Identification and Measurement Protocol]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: Flow Cytometry Calibration Protocols

Flow cytometers must be calibrated prior to fluorescence intensity measurements because of inherent instrument variability. To correct for this variability, a standard particle (fixed chicken red blood cells, or CRBCs) must be analyzed on the instrument prior to each experiment and photomultiplier tube (PMT) voltages adjusted accordingly to place the CRBC fluorescence emission peaks into predetermined target channels. - [Read Calibration of Becton Dickinson Flow Cytometers for Relative Fluorescence Intensity Measurements]

Category: Microscopy Protocols: Live-Cell Imaging Protocols

Manual measurement and manipulation of the cell surface requires access to the cells, usually in an open chamber. Temperature-controlled chambers or stage inserts are preferred for maintaining physiological activity during the experiment. For example, heated culture dishes with coverslip glass bottoms (Bioptechs) permit high-resolution fluorescence microscopy of living cells during force application. - [Read Chambers for Examination of Live Cells under Mechanical Stress Protocol]

Category: Cytogenetics Protocols: In Situ Hybridization Protocols

Choosing the right labeling method for your hybridization experiment. Includes: Homogeneous labeling methods for DNA; Homogeneous labeling methods for RNA; Stability of probe-target interaction; Nonradioactive labeling of oligonucleotides; Double-stranded versus single-stranded probes. - [Read Choosing the Right Labeling Method for your Hybridization Experiment]

Category: PCR Protocols: Reverse Transcriptase RT-PCR Protocols

In this protocol, sample and competitor RNAs are reverse transcribed (separately) in a pilot experiment.  A constant amount of sample RT product is then combined with a 2-logserial dilution of competitor RT product for PCR. Procedure provides an approximate copy number for the sample, which is then fine-tuned by repeating the experiment with a series of twofold dilutions of competitor.  The experiment includes controls for sample-to-sample variations in RT efficiency. - [Read Competitive RT-PCR: Estimation of Copy Number Protocol]

Category: Cytogenetics Protocols

CGH provides a comprehensive picture of all chromosomal gains and losses within a single experiment. In contrast to banding analysis, CGH requires no preparation of metaphase chromosomes from the cells to be analyzed (which in certain tumor types may be difficult or even impossible). - [Read Detection of Chromosomal Imbalances Using DOP-PCR and Comparative Genomic Hybridization (CGH)]

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: Immunology: Mononuclear Cell Isolation Protocols

This protocol uses the PBMC fraction enriched in with monocytes by density gradient centrifugations (protocol may be found at www.methods.info). Reduction of the amount of microbeads in comparison to Miltenyi protocol reduces the costs of the experiment. - [Read Isolation of Monocytes from Enriched PBMCs using CD14 Magnetic Beads Protocol]

Category: Cell Biology and Cell Culture

This protocol uses the PBMC fraction enriched in with monocytes by density gradient centrifugations. Reduction of the amount of microbeads reduces the costs of the experiment. - [Read Isolation of monocytes from monocyte enriched PBMC fraction using CD14]

Category: DNA Microarray Protocols

The "7 keys to successful microarray analysis" has been designed to guide researchers step-by-step through the design, implementation, analysis, and publication of a microarray experiment. - [Read Microarray Success]

Category: Neuroscience Protocols: Neuronal Cell Culture Protocols

Neuron-fibroblast coculture assay to demonstrate presynaptic differentiation.  Includes: Preparation of HEK293T cells, Coculture of HEK293T cells and neurons, Immunostaining, Vesicle turnover experiment, Image acquisition and quantitation. - [Read Neuron Fibroblast Coculture Assay to Demonstrate Presynaptic Differentiation]

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols: Genomic DNA Isolation Protocols

The results of the pilot experiment (Partial Digestion of Eukaryotic DNA for Use in Genomic Libraries: Pilot Reactions) are used to establish conditions for partial digestion of eukaryotic DNA in the large-scale reactions described here. - [Read Partial Digestion of Eukaryotic DNA for Use in Genomic Libraries: Preparative Reactions Protocol]

Category: Plant Biology Protocols: Arabidopsis Protein Peptide Protocols

Coimmunoprecipitation is one of the most useful techniques for revealing protein-protein interactions. Good negative controls to verify the specificity of the coimmunoprecipitation procedure are (1) performing the same immunoprecipitation experiment using beads coupled to the preimmune serum and (2) probing the Western blot with antibodies against protein known not to interact with the coimmunoprecipitated proteins under physiological conditions. - [Read Protein Coimmunoprecipitation in Arabidopsis Protocol]

Category: Nucleic Acid Detection Protocols

Restriction landmark genomic scanning (RLGS) is a method to detect large numbers of restriction landmarks in a single experiment.  It is based on the concept that restriction enzyme sites can serve as landmarks throughout a genome.  RLGS uses direct end-labeling of the genomic DNA digested with a rare-cutting restriction enzyme and high-resolution two-dimensional electrophoresis. - [Read Restriction Landmark Genomic Scanning Protocol]

Category: Chromatography Protocols: Ion Exchange Chromatography Protocols

Protocol for separating proteins using ion-exchange chromatography. Protocol details the practical considerations of an ion-exchange chromatography (IEC) experiment.  The choice of what type of ion exchanger to use, as well as the composition of the buffers used in this experiment, should be determined prior to beginning this protocol. - [Read Separating Proteins Using Ion-Exchange Chromatography Protocol]

Category: Stem Cell Protocols: Stem Cell Culture Protocols

Protocol describes how to set up microdrop cultures to produce embryos which can then be used for making chimeras. The microdrop culture should be set up several hours to 1 day before the experiment to permit temperature and gas equilibration. - [Read Setting Up Microdrop Cultures Protocol]