mediated Protocols

Category: PCR Protocols: cDNA Synthesis Protocols

New RACE, a variation of RNA ligase-mediated-RACE (RLM-RACE) (Liu and Gorovsky 1993) departs from classic RACE (see 5'-End cDNA Amplification Using Classic RACE) in that an "anchor" primer is attached to the 5'-end of the mRNA before the reverse transcription step; hence the anchor sequence becomes incorporated into the first-strand cDNA if, and only if, the reverse transcription proceeds through the entire length of the mRNA of interest. - [Read 5'-End cDNA Amplification Using New RACE Protocol]

Category: Plant Biology Protocols: Plant Genetics Protocols

Protocol describing Agrobacterium mediated gene transfer via hypocotyls. - [Read Agrobacterium-Mediated Gene Transfer via Hypocotyls Protocol]

Category: Cytogenetics Protocols

Protocol for Agrobacterium-mediated transformation in rice. Agrobacterium-mediated rice transformation method that is applicable to easily cultured varieties in addition to elite japonica varieties that are more difficult to culture. Using this method, transgenic rice plants can be obtained in about 2–3 months with a transformation frequency of 30–50%, both in easily cultured varieties and recalcitrant elite japonica rice. - [Read Agrobacterium-Mediated Transformation in Rice Protocol]

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: C. elegans Protocols

Protocol for bacteria- mediated (feeding) RNAi. - [Read Bacteria-Mediated (Feeding) RNAi Protocol]

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: Transfection Protocols: Calcium Phosphate Transfection Protocols

Calcium phosphate forms an insoluble precipitate with DNA, which attaches to the cell surface and is taken into the cells by endocytosis. The protocol is easily adapted for use with other types of cells, both adherent and nonadherent. This protocol is a modified version of a method published by Jordan et al. (1996) who rigorously optimized calcium-phosphate-based transfection methods for Chinese hamster ovary cells and the 293 line of human embryonic kidney cells. - [Read Calcium-phosphate-mediated Transfection of Eukaryotic Cells with Plasmid DNAs]

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

Because sequencing reactions catalyzed by thermostable DNA polymerases such as Taq are carried out at elevated temperatures, problems caused by mismatched annealing of primers or templates rich in secondary structure are greatly alleviated. - [Read Dideoxy-mediated Sequencing of DNA Using Taq DNA Polymerase Protocol]

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

Protocol describes DNA-sequencing of single-stranded DNA templates in reactions catalyzed by Sequenase. - [Read Dideoxy-mediated Sequencing Reactions Using Bacteriophage T7 DNA Polymerase (Sequenase) Protocol]

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

Protocol for dideoxy-mediated sequencing reactions using the Klenow fragment of E. coli DNA polymerase I and single-stranded DNA templates. - [Read Dideoxy-mediated Sequencing Reactions Using the Klenow Fragment of E. coli DNA Polymerase]

Category: E Coli Protocols: E coli Nucleic Acid Protocols

Protocol for dideoxy-mediated sequencing reactions using the Klenow Fragment of E. coli DNA polymerase I and single-stranded DNA templates. - [Read Dideoxy-mediated Sequencing Reactions Using the Klenow Fragment of E. coli DNA Polymerase I]

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: Cell Biology and Cell Culture: In Vitro Reconstitution Protocols

This protocol describes the use of SEMV cells (a cell line derived from ram seminal vesicles) in studies into prostaglandin H synthase-mediated metabolism of xenobiotics in intact cells. - [Read In Vitro Model For Studies Of Prostagland H Synthase (PHS)- Mediated Genotoxicity Of Xenobiotics]

Category: Fungi Protocols: Neurospora Protocols

Lipofectin increases the efficiency of DNA-mediated transformation of Neurospora crassa. - [Read Lipofectin Increases the Efficiency of DNA-Mediated Transformation of Neurospora Crassa]

Category: PCR Protocols

DNA prepared by PCR-mediated gene disruption can be used to transform yeast in gene replacement experiments.  This protocol uses two primers, tailed with approximately 50 nucleotides homologous to the gene of interest, that target insertion of the PCR product to that locus.  Each primer ends with a universal sequence that is designed to amplify various selectable markers from plasmid templates. - [Read PCR-Mediated Gene Disruption: One-Step Method Protocol]

Category: Developmental Biology: Utilizing Model Organisms Protocols

Nonviral, DNA-mediated gene transfer is an alternative to viral delivery systems for expressing new genes in cells and tissues. The Sleeping Beauty (SB) transposon system combines the advantages of viruses and naked DNA molecules for gene therapy purposes; however, efficacious delivery of DNA molecules to animal tissues can still be problematic. Here we describe the hydrodynamic delivery procedure for the SB transposon system that allows efficient delivery to the liver in the mouse. - [Read Preferential Delivery of the Sleeping Beauty transposon System to Livers of Mice by Hydrodynamic i]

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

Protocol describes how double-stranded plasmid DNA is denatured by alkali and annealed to an appropriate oligonucleotide primer, in preparation for dideoxy-sequencing reactions catalyzed by Sequenase. - [Read Preparing Denatured Templates for Sequencing by Dideoxy-mediated Chain Termination Protocol]

Category: RNA Protocols: RT-PCR and cDNA synthesis

This procedure was first described by Bertrand et al to demonstrate that ribozymes could be enzymatically active in vivo. We adapted the method to show that certain oligodeoxynucleotides could direct the activity of endogenous ribonuclease H to cleave tar - [Read Reverse Ligation Mediated RT - PCR]

Category: Plant Biology Protocols: Arabidopsis Transformation Protocols

Arabidopsis can be stably transformed using Agrobacterium tumefaciens-mediated transfer of T-DNA. We describe the generation of transgenic plants via root transformation in tissue culture, which can be useful for transforming sterile mutants. - [Read Root Transformation of Arabidopsis Protocol]

Category: Plant Biology Protocols

Protocol includes: Seed Sterilization Step; Seed Germination Step; Agrobacterium Inoculum Preparation; Explant Preparation; Co-cultivation Plates; Shoot Initiation Step; Shoot Elongation Step; Rooting Step. - [Read Soybean Cotyledonary-node Agrobacterium-mediated Transformation System]

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

GUS is used as a tag to address nuclear localization whereas GFP is more versatile. GFP is detectable directly in living cells, GUS is only detected indirectly by staining of fixed tissue which may lead to artifacts or may obscure problems with protein solubility. In this protocol, protein localization is routinely assayed after particle-mediated transient transformation of onion epidermal cells. With this method it can be determined rapidly whether a given fusion protein is active and.... - [Read Subcellular Localization of GUS- and GFP-Tagged Proteins in Onion Epidermal Cells]

Category: Transfection Protocols

DEAE-dextran is generally used to obtain a burst of transient expression of cloned genes after transfection of mammalian cells. Many variants of the technique have been described, all of which seek to maximize the uptake of DNA and to minimize the cytotoxic effects of DEAE-dextran. In this protocol cells are exposed briefly to a high concentration of DEAE-dextran-DNA and then to chloroquine diphosphate, which is a facilitator of transfection. - [Read Transfection Mediated by DEAE-Dextran: High-efficiency Method Protocol]

Category: Plant Biology Protocols: Plant Genetics Protocols

The study of transient gene expression provides a useful complement to the study of stably transformed plants. Transient assays offer a quick method of testing the effects of genes, using either phenotypic, molecular, or biochemical readouts. Transient assays based on Agrobacterium-mediated transformation of leaf explants have been described for other plant species, but it is not known how well these assays work in Arabidopsis. - [Read Transient Expression in Protoplasts]

Category: Cloning Protocols: Mutagenesis Protocols

Protocol for transposon mediated mutagenesis. Includes: Amplify ORF from MG1655; Transposition reaction; Transformation; Picking; Verify mutation by Culture PCR; Confirm mutations by sequencing; Curing the temperature sensitive pKD46 plasmid. - [Read Transposon Mediated Mutagenesis Protocol]