involves Protocols

Category: PCR Protocols: AFLP PCR

An introduction to AFLP and fAFLP. Mark E. Berres, University of Wisconsin. Amplified fragment-length polymorphism (AFLP) or its fluorescent version (fAFLP) is a PCR-based fingerprinting technology. AFLP basically involves the restriction of genomic DNA - [Read An introduction to AFLP and fAFLP]

Category: Transfection Protocols: Stable Transfection Protocols

The AfCS is utilizing antisense technology to manipulate signaling protein expression in the RAW 264.7 macrophage-like cell line. This can be achieved by the transfection of gene-specific antisense oligonucleotides (ASOs). The following procedure involves the transfection of ASOs into RAW 264.7 cells using FuGENE 6 transfection reagent. Subsequently, the isolated total RNA or protein from these transfected cells can be used to assess the level of mRNA or protein knockdown, respectively. - [Read Antisense Oligonucleotide Transfection of RAW 264.7 Cells with FuGENE 6 in a 24-Well Dish]

Category: Protein Protocols: Protein Quantitation Concentration Protocols: Bradford Protein Assay Protocols

The bradford dye-binding assay is a colorimetric assay for measuring total protein concentration.  It involves the binding of Coomassie Brilliant blue to protein. There is no interference from cations nor from carbohydrates such as sucrose. 
Detergents such as sodium dodecyl sulfate and triton x-100 can interfere with the assay, as well as strongly alkaline solutions.

Includes a general overview of the procedure and preparation of the standards in the protocol. - [Read Bradford Assay Method]

Category: PCR Protocols: cDNA Synthesis Protocols

This method, for the selective amplification of full-length cDNA ends, involves the addition of an adapter during reverse transcription.  This method takes advantage of the propensity of Moloney murine leukemia virus reverse transcriptase (MMLV RT) to append two to four cytosines to the 3'-end of newly synthesized cDNA strands.  The additional residues are added when the enzyme reaches the 5'-cap structure at the end of the mRNA template. - [Read Cap-Switching RACE Protocol]

Category: Cell Biology and Cell Culture: Cell Viability Protocols

The CellTiter-Blue® Cell Viability Assay uses an optimized reagent containing resazurin. The homogeneous procedure involves adding the reagent directly to cells in culture at a recommended ratio of 20µl of reagent to 100µl of culture medium. - [Read Cell Viability Assays that Measure Metabolic Capacity Protocol]

Category: Immunology: Immunoprecipitation Protocols: Immunoprecipitation of Radiolabeled Cells Protocols

Most powerful and convincing method to determine if a specific protein is phosphorylated in a physiologically relevant manner is to assay phosphorylation in situ. The procedure described involves incubating cultured cells (e.g., primary neuronal cultures or transfected cells) or tissue preparations (e.g., hippocampal slices) with [32P]orthophosphate, which is then taken up by the cells or tissues and incorporated into the γ-phosphate position of ATP. - [Read Detection of Protein Phosphorylation in Tissues and Cells Protocol]

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: DNA Protocols: cDNA Library Protocols: cDNA Library Construction Protocols

Shotgun sequencing of a large segment of DNA involves random fragmentation of the target region into smaller segments that are subsequently cloned into a bacteriophage M13 vector. The goal is to create a library of overlapping clones that provide at least fivefold coverage over the entire length of the target fragment. - [Read Generation of a Library of Randomly Overlapping DNA Inserts Protocol]

Category: Primary Cell Isolation and Culture Protocols: Laser Capture Microdissection Protocols

Useful techniques to circumvent disruption of tissue structure in the analysis of gene expression are LCM and LDM. While they require specialized microscopes and systems, they are similar in that freshly-cut frozen tissue sections can be microdissected using either a general histological stain (like H&E) or by staining with fluorescently conjugated antibodies. The LCM system by Arcturus involves... - [Read Immunofluorescent Staining for the Laser Microdissection of Individual Cells Protocol]

Category: PCR Protocols: Inverse PCR Protocols

Inverse PCR is used to amplify and clone unknown DNA that flanks one end of a known DNA sequence and for which no primers are available.  The technique involves digestion by a restriction enzyme of a preparation of DNA containing the known sequence and its flanking region.  The individual restriction fragments (many thousands in the case of total mammalian genomic DNA) are converted into circles by intramolecular ligation, and the circularized DNA is then used as a template in the PCR. - [Read Inverse PCR Protocol II]

Category: Immunology: Mononuclear Cell Isolation Protocols

Presents two methods for preparing dendritic cells (DCs), a highly specialized type of antigen-presenting cell (APC). The first method involves the isolation of DCs from mouse spleen, resulting in a cell population that is highly enriched in accessory cell and APC function. A support protocol for collagenase digestion of splenocyte suspensions is described to increase the yield of dendritic cells. The second method involves generating large numbers of DCs from mouse bone marrow progenitor cells. - [Read Isolation of Dendritic Cells Protocol]

Category: Antibody Protocols: Antibody Labeling Protocols

This method for tagging monoclonal antibodies involves growing hybridomas in the presence of radioactive amino acids. This protocol can be particularly useful when conventional labeling techniques cause the antibody to lose activity. The labeled antibodies that result are essentially identical to the unlabeled antibodies. - [Read Labeling Monoclonal Antibodies by Biosynthesis Protocol]

Category: DNA Protocols: cDNA Library Protocols

The final stage of cDNA library construction involves optimizing ligation of the size-fractionated cDNA to bacteriophage {lambda} arms, followed by packaging and plating of the recombinant bacteriophages. - [Read Ligation of cDNA to Bacteriophage lambda Arms for the Construction of cDNA Libraries]

Category: Plant Biology Protocols

The standard protocol for in situ hybridizations in plants still involves fixing fresh tissue, embedding the tissue in wax, sectioning with a microtome and detection of the transcripts of interest using labeled RNA-probes. This protocol concentrates only on nonradioactive methods, as they are easy to perform, very sensitive and even faster than techniques involving radioisotope labels. - [Read Molecular and Biochemical Analysis of Arabidopsis Protocol]

Category: Cytoskeleton Protocols: Microtubules Protocols

Negative staining is a rapid, qualitative method for analyzing microtubule structure at the EM level. Because negative staining involves deposition of heavy atom stains, structural artifacts such as flattening of the cylindrical microtubule and opening up of microtubules into flat sheets are common. Negative staining is very useful because of its ease, rapidity and lack of requirement for specialized equipment other than that found in a regular EM facility. - [Read Negative Stain Electron Microscopy of Microtubules Protocol]

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

A procedure developed to purify simultaneously peroxisomes and mitochondria from spinach (Spirnacia olercea L.) leaf under isoosmotic and low viscosity conditions. This method involves differential centrifugation and density gradient centrifugation on four layers of Percoll. - [Read Purification of Peroxisomes and Mitochondria from Spinach Leaf by Percoll Gradient Centrifugation]

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

A procedure developed to purify simultaneously peroxisomes and mitochondria from spinach (Spirnacia olercea L.) leaf under isoosmotic and low viscosity conditions. This method involves differential centrifugation and density gradient centrifugation on four layers of Percoll. - [Read Purification of Peroxisomes and Mitochondria from Spinach Leaf by Percoll Gradient Centrifugation]

Category: Nucleic Acid Modification Protocols: DNA Methylation Protocols

Protocol for quantification of DNA methylation in electrofluidics chips. Describe Bio-COBRA, a modified protocol for Combined Bisulfite Restriction Analysis (COBRA), that incorporates an electrophoresis step in microfluidics chips.  Microfluidics technology involves the handling of small amounts of liquid in miniaturized systems. - [Read Quantification of DNA Methylation in Electrofluidics Chips Protocol]

Category: PCR Protocols: Quantitative PCR Protocols

Quantitative PCR involves co-amplification of two templates: a constant amount of a preparation containing the desired target sequence and serial dilutions of a reference template that is added in known amounts to a series of amplification reactions.  The concentration of the target sequence is determined by simple interpolation into a standard curve. - [Read Quantitative PCR Protocol II]

Category: RNAi Technology Protocols

Protocol Involves the transfection of siRNA into RAW 264. 7 cells using Lipofectamine 2000.  Cells are transfected with siRNA twice (on subsequent days).  Target gene knockdown is assessed from total RNA isolated 48 hr post-transfection or from protein isolated 72 hr post-transfection. - [Read siRNA Double Transfection of RAW 264.7 Cells with Lipofectamine Protocol]

Category: RNAi Technology Protocols

Protocol Involves the transfection of siRNA into RAW 264. 7 cells using Lipofectamine 2000.  Cells are transfected with siRNA twice (on subsequent days).  Target gene knockdown is assessed from total RNA isolated 48 hr post-transfection or from protein isolated 72 hr post-transfection. - [Read siRNA Double Transfection of RAW 264.7 Cells with Lipofectamine Protocol II]