generate Protocols

Category: PCR Protocols: cDNA Synthesis Protocols

To generate "3'-end" partial cDNA clones, mRNA is reverse-transcribed using a "hybrid" primer (Qtotal, QT) that consists of two mixed bases (GATC/GAC followed by [T]17) and a unique 35-base oligonucleotide sequence (QI-QO).  Amplification is then performed using a primer containing part of this sequence (Qouter, Qo) (which now binds to each cDNA at its 3'-end) and a primer derived from the gene of interest, GSP1 (gene-specific primer 1). - [Read 3'-End cDNA Amplification Using Classic RACE Protocol]

Category: PCR Protocols: cDNA Synthesis Protocols

To generate "5'-end" partial cDNA clones using classic RACE, the first-strand products are generated by reverse transcription (primer extension) from a known gene-specific primer (GSP-RT).  Then, a poly(A) tail is appended using terminal deoxynucleotidyltransferase (Tdt) and dATP.  Amplification is carried out using three primers. - [Read 5'-End cDNA Amplification Using Classic RACE Protocol]

Category: Yeast Protocols: Yeast Nucleic Acid Protocols: Yeast PCR Protocols

Protocol for 96-well confirmation Yeast PCR. Includes: Clonal purification; Generate a master plate (96-well format); Making a frozen backup stock; Confirmation PCR for one Row; ORF Specific Confirmation PCR --> "A-B" primers (upstream junction); Transfer template DNA to multiwell PCR plate; Prepare and dispense master mix for A-B PCR. - [Read 96-Well Confirmation Yeast PCR Protocol]

Category: Plant Biology Protocols: Plant Genetics Protocols

AFLP was designed as a highly sensitive method for DNA fingerprinting to be used in a variety of fields. We are using this technology to generate DNA based markers for cloning genes involved in phototropic responses in higher plants that have only been identified genetically by mutant phenotype. Protocol includes: Generate polymorphic recombinant F2 (or F3) population; Isolate genomic DNA; Restriction of DNA; Ligation of adapters; Pre-amplification of template DNA; AFLP-PCR; etc. - [Read AFLP For Positional Cloning]

Category: PCR Protocols: AFLP PCR

Mannie Liscum and Paul Oeller. Department of Plant Biology. Carnegie Institution of Washington, Stanford. AFLP technology is used here to generate DNA based markers for cloning genes involved in phototropic responses in higher plants that have only been i - [Read AFLP: not only for fingerprinting, but for positional cloning]

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

Analysis of DNA Fragmentation Using the JAM Assay. By Shailaja Kasibhatla et al., The JAM assay is based on labeling nuclear DNA of cycling cells with [3H]thymidine and harvesting samples on glass fiber filters. Apoptosis will generate DNA fragments small enough to pass through the glass fiber filter, resulting in decreased radioactivity of the particular sample. Cell-mediated cytotoxicity or cell killing mediated by cytotoxic T lymphocytes (CTL) can also be measured by this technique. - [Read Analysis Of DNA Fragmentation Using The JAM Assay (Subscription Required)]

Category: Chromatography Protocols: Immunoaffinity Chromatography Protocols

ANTIBODY PURIFICATION by affinity chromatography. By Beth, Mullins Lab UCSF. To affinity purify antibodies, generate lots of E. coli lysate that contains your antigen. If the protein can stand freeze thawing, then go ahead and purify the protein from e. coli lysate and keep it frozen until you need to couple it to a CH-sepharose column. - [Read ANTIBODY PURIFICATION by affinity chromatography]

Category: Transfection Protocols: Calcium Phosphate Transfection Protocols

This protocol,is based on the venerable method of Graham and van der Eb (1973). The procedure is used chiefly to generate stable lines of cells carrying chromosomally integrated copies of the transfected DNA. - [Read Calcium-phosphate-mediated Transfection of Cells with High-molecular-weight Genomic DNA]

Category: DNA Protocols: DNA Sequencing Protocols: Dideoxy Mediated Sequencing Protocols

In this protocol asymmetric PCR is used to generate single-stranded DNA templates for dideoxy-sequencing - [Read Dideoxy-mediated Sequencing Reactions Using PCR and End-labeled Primers Protocol]

Category: Stem Cell Protocols

Pluripotent ES cells can develop into many types of differentiated tissues if they are placed back into a differentiating environment. Often, differentiation proceeds through an intermediate stage called the embryoid body (EB). EBs can be manipulated further to generate more differentiated cell types. This protocol describes a method for differentiation of ES cells into EBs. - [Read Differentiating Embryonic Stem (ES) Cells into Embryoid Bodies Protocol]

Category: Cloning Protocols: Vector Protocols

Directional cloning requires that the plasmid vector be cleaved with two restriction enzymes that generate incompatible termini and that the fragment of DNA to be cloned carries termini that are compatible with those of the doubly cleaved vector. - [Read Directional Cloning into Plasmid Vectors Protocol]

Category: Genetics and Genomics: Genotyping Protocols: Mutation Detection Protocols

The double-stranded DNA of recombinant plasmid, phagemid, or bacteriophage M13 replicative form DNA is digested with two restriction enzymes whose sites of cleavage both lie between one end of the target DNA and the binding site for universal primer. The enzyme that cleaves nearer the target sequence must generate either a blunt end or a recessed 3' terminus; the other enzyme must generate a four-nucleotide protruding 3' terminus. - [Read Generation of Sets of Nested Deletion Mutants with Exonuclease III Protocol]

Category: Viral Manipulation Protocols: M13 Phage Manipulation Protocols

Protocol used chiefly to generate large stocks of double-stranded DNA of strains of M13 that are routinely used as cloning vectors. Large amounts of single-stranded DNA of an individual recombinant may occasionally be needed for specific purposes, e.g., to generate many preparations of a particular radiolabeled probe or to construct large numbers of site-directed mutants. - [Read Large-scale Preparation of Single-stranded and Double-stranded Bacteriophage M13 DNA Protocol]

Category: Bacterial Protocols

Ice tea has a complex composition, which leads to reduced filterability, and a decrease in sample throughput. Its composition can generate background or false positive signals. It is also well known that ice tea contains molecules that can inhibit the bioluminescence reaction, which can generate false negative results. The aim of this study was to develop a protocol that was able to neutralize these affects and enable faster detection of contamination. - [Read Microbial Detection in Ice Tea Using the Millipore Milliflex Rapid Microbiology Detection System]

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

Protocol is used to establish conditions for restriction enzyme digestion of eukaryotic genomic DNA that will generate fragments of a size appropriate for construction of genomic libraries.  To construct a genomic library, the average length of the starting genomic DNA should be at least eight times the capacity of the vector. - [Read Partial Digestion of Eukaryotic DNA for Use in Genomic Libraries: Pilot Reactions Protocol]

Category: Molecular Biology Protocols: Phage Protocols and Methods

Plating Lambda Phage to Generate Plaques. Elution of Phage. Phage DNA Extraction. Dr.Wurtzel. Dept. Biological Sciences, Lehman College. - [Read Phage Titer]

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: C. elegans Protocols

Protocol was designed to rapidly generate small scale cytosolic extracts of C. elegans for Western or IP (has not been tested for RNA work). The protocol works well for between 50 to 5000 worms and has not been extensively tested on larger a scale, though it should work. Includes: Collection; Sonication; Clearing lysate; Immunoprecipitation. - [Read Preparation of Worm Extracts by Sonication Protocol]

Category: Developmental Biology: Utilizing Model Organisms Protocols

Protocols on the genetics of Pristionchus pacificus. Includes: Freezing worms; Mutagenesis; Construction of deletion libraries to generate P. pacificus gene knock-outs; RNAi and morpholino by injection. - [Read Pristionchus Pacificus Genetic Protocols]

Category: Laboratory Model Organisms Protocols

Genetic Protocols for Pristionchus pacificus. Includes: Freezing worms; EMS mutagenesis; Psoralen mutagenesis; Construction of deletion libraries to generate P. pacificus gene knock-outs; Designing primers for the gene of interest; RNAi and morpholino by injection. - [Read Pristionchus pacificus Genetic Protocols]

Category: Immunology: T Cell Protocols

In the first protocol, IL-2-producing murine T cells are measured following stimulation by the mitogen Con A. The second protocol provides a modification for using human responder cells. The second protocol is used for estimating the proportion of cells that can generate a clone of cytotoxic effector cells when stimulated by Con A with the addition of IL-2. - [Read Quantitation of Functional T Cells by Limiting Dilution Protocols]

Category: Genetics and Genomics: Cancer Genetics Protocols

SAGE is a new method that has been invented at Johns Hopkins University in USA to give scientists an overview of a cell’s complete gene activity. It works by capturing RNAs, identifying them and counting them. By comparing different types of cells, the researchers hope to generate profiles that will help them understand healthy cells and what goes wrong during diseases. Includes: How SAGE works and Steps of SAGE. - [Read Serial Analysis Of Gene Expression (SAGE)]

Category: Transfection Protocols: Stable Transfection Protocols

Protocol uses an autoregulatory system in which the transcriptional trans-activator tTA drives its own expression and that of a target gene. The first stage of the procedure describes how to generate stable lines of NIH-3T3 cells that express either tTA alone or tTA and the tetracycline-regulated target gene. - [Read Tetracycline as Regulator of Inducible Gene Expression Protocol]

Category: Yeast Protocols: Yeast Two-Hybrid Systems Protocols

Protocol describes how to generate a plasmid construct (pBAIT) that expresses a target protein fused to the bacterial LexA protein. PBAIT is cotransformed into yeast with a lexAop-lacZ reporter plasmid carrying the bacterial lacZ gene under the control of the lexA operator. The recipient yeast strain contains a chromosomally integrated leu2 reporter gene, also under the control of the lexA operator. - [Read Two-hybrid Systems Stage 1: Characterization of a Bait-LexA Fusion Protein Protocol]

Category: Viral Culture and Isolation Protocols: Viral Isolation Protocols

Use of “293-GPG” cells to generate amphotropic retroviruses, plus infection protocol. - [Read Use of “293-GPG” cells to Generate Amphotropic Retroviruses, plus Infection Protocol]