direct Protocols

Category: BACs Bacterial Artificial Chromosome Protocols

Protocol presents the amplification of insert end sequences from bacterial artificial chromosome clones using TAIL-PCR. The amplified products are suitable as probes for chromosome walking and genome mapping and as templates for direct sequencing. The protocol has been used in rice genome studies. - [Read Amplification of Insert End Sequences from BACs Clones by Thermal Asymmetric Interlaced PCR]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols

A sensitive method for the detection of apoptosis by single laser flow cytometry. Methodology includes: Staining for detection of apoptosis, Direct Staining Procedure, Indirect Staining Procedure, Protocol for the use of actinomycin D (AD) on samples that were stained with 7-AAD for apoptosis and fixed in formaldehyde. - [Read Apoptosis Detection Protocol By Single Laser Flow Cytometry]

Category: Cell Biology and Cell Culture

Protocol applies EFs to cells in vitro but has been modified and to use electrotactic chambers to accommodate cells growing in planar culture or in three-dimensional (3D) gels, en bloc tissue cultures in 3D and possible small embryos, such as that from frog and zebra fish. The EF is applied to the cells or tissues cultured in a customer designed electrotactic chamber via agar salt bridges, Steinberg’s solution and Ag/AgCl electrodes. - [Read Application of Direct Current Electric Fields to Cells and Tissues in vitro]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols

Basic Method for Direct Immunofluorescence Labeling. Iowa State University, Flow Cytometry Facility. - [Read Basic Method for Direct Immunofluorescence Labeling]

Category: PCR Protocols: Bisulfite Conversion Based PCR Protocols

Protocol for bisulfite-PCR for restriction analysis and/or sequencing. Bisulfite-PCR followed by restriction is a rapid and semi-quantitative method of analyzing DNA methylation.  The PCR products are also suitable for either direct sequencing or cloning and sequencing.  The most important step here is primer selection. - [Read Bisulfite-PCR for Restriction Analysis and/or Sequencing Protocol]

Category: Cytogenetics Protocols

This CGH Protocol is used for DNA of good quality when available in sufficient amounts. We usually do replicate hybridizations using samples labeled "inversely" (reversing the label for test and normal DNA's). If appreciable artifact occurs, then alternative labels are tried. - [Read CGH of Direct Labeled Test DNA vs Normal DNA Protocol]

Category: Cytogenetics Protocols: DIG Biotin Labeled Probe Protocols

This protocol describes how to use DIG Chem-Link to directly label any DNA [e.g. plasmids, PCR products, cDNA prepared from mRNA] or RNA (e.g. total RNA, poly(A)+ mMRNA). The DIG Chem-Link or Biotin Chem-Link may also be used to label oligonucleotides. Includes: Required Purity of DIG Chem-Link Templates; Direct DIG Labeling of mRNA or cDNA with DIG Chem-Link; Key Product Required for Direct Labeling of DNA or RNA; Estimating the Yield of DIG-labeled Nucleic Acids. - [Read Chem-Link Labeling of DNA or RNA with DIG or Biotin Protocol]

Category: Nucleic Acid Modification Protocols: DIG Biotin Labeled Probes Protocols

Protocol for Chem-Link labeling of DNA or RNA with DIG or Biotin. Includes: Direct DIG Labeling of mRNA or cDNA with DIG Chem-Link; Estimating the Yield of DIG-labeled Nucleic Acids. - [Read Chem-Link Labeling of DNA or RNA with DIG or Biotin Protocol]

Category: Cell Biology and Cell Culture

This chemotaxis assay protocol is based on the premise of creating a gradient of the chemotactic agent and allowing cells to migrate through a membrane towards the chemotactic agent. A chemotaxis assay can determine whether your protein or small molecule of interest has chemotactic activity on a specific cell type. Chemotaxis is then the ability of a protein to direct the migration of a specific cell. - [Read Chemotaxis Assay Protocol]

Category: PCR Protocols: PCR Cloning Protocols

This method of direct cloning takes advantage of the unpaired adenosyl residue added to the 3' terminus of amplified DNAs by Taq and other thermostable polymerases. - [Read Cloning PCR Products into T Vectors Protocol]

Category: E Coli Protocols: E coli Genetics Protocols

Protocol for Colorimetric Assay to Identify Putative Ribofuranosylaminobenzene 5'-Phosphate Synthase Genes. The production of active RFAP synthase from Methanothermobacter thermautotrophicus was achieved by coexpression of the gene MTH0830 with a molecular chaperone. This is the first direct biochemical identification of a methanogen gene that codes for an active RFAP synthase. - [Read Colorimetric Assay to Identify Putative Ribofuranosylaminobenzene 5'-Phosphate Synthase Genes]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: Cell Cycle Analysis Protocols

Describes assays used to determine the distribution of a population of cells to the different stages of the cell cycle as analyzed by flow cytometry. Staining the DNA with different fluorescent dyes, propidium iodide or DAPI, is one of the most direct ways of staging the cells based on DNA content. - [Read Determining Cell Cycle Stages by Flow Cytometry Protocol]

Category: Yeast Cell Culture Protocols: Yeast Sporulation Protocols

Direct method for determining efficiency in yeast. The plating efficiency of a strain is a measure of the percentage of cells in a culture that are capable of forming colonies (colony forming . - [Read Determining Plating Efficiency in Yeast: Direct Method]

Category: Cell Culture Protocols: Insect Cell Culture Protocols

Using this method, insect cells should be adapted to HyQ SFX-Insect in 4-8 passages. However, there are some cell lines and clones that are more sensitive to the physiochemical and nutritional changes, and in some cases it may take longer than 8 passages for the adaptation. - [Read Direct Adaptation of Insect Cells to HyQ© SFX-Insect™ Medium]

Category: ELISA Protocols

Direct Elisa Using Fluorescent Substrate Protocol. Abcam. - [Read Direct Elisa Using Fluorescent Substrate Protocol]

Category: ELISA Protocols

Direct Elisa Using Primary Antibody Protocol. Abcam. - [Read Direct Elisa Using Primary Antibody Protocol]

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

Protocol for direct PCR from whole Yeast cells. - [Read Direct PCR from Whole Yeast Cells Protocol]

Category: Nucleic Acid Electrophoresis Protocols: Pulsed Field Gel Electrophoresis Protocols

Protocol for direct retrieval of DNA fragments from pulsed-field gels. A gel slice containing a fragment of DNA resolved by pulsed-field gel electrophoresis is treated with agarase.  The released DNA can be used as a substrate for ligation or restriction without further purification. - [Read Direct Retrieval of DNA Fragments from Pulsed-field Gels Protocol]

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

The goal of this method is to identify transcriptionally active genes in cloned segments of genomic DNA. The protocol uses hybridization and affinity purification to recover biotin-labeled cDNAs that bind to a 500-kb segment of human DNA cloned in a BAC vector. However, the method can be easily adapted to other clones of genomic DNAs cloned in high-capacity vectors. - [Read Direct Selection of cDNAs with Large Genomic DNA Clones Protocol]

Category: DNA Protocols: DNA Sequencing Protocols

Direct sequencing from amplified bacterial and large insert cloned human or mouse genomic DNA via an improved MultiPlex PCR-based method. Includes: Preparing primers for MP-PCR; Amplification; PCR Product Clean-up; Sequencing. - [Read Direct Sequencing Using MultiPlex PCR-Based Method]

Category: Immunology: B Cell Protocols

B cell activation can be quantitated indirectly by assaying antibody production or directly by measuring cellular changes that occur immediately after exposure to an activation signal. Provides methods for the latter (direct) approach--namely, methods for quantifying early parameters of B cell activation such as increases in intracellular ionized calcium concentration [Ca2+]I, cell size, and MHC class II-antigen expression. - [Read Early Events in B Lymphocyte Activation Protocol]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: Flow Cytometry Immunofluorescence Staining Protocols

Protocol provides a method for analysis of cell surface antigens using direct immunofluorescence, i.e., when the antibody being used is directly labeled to a fluorescent dye. After staining, the cells can be analyzed immediately by flow cytometry or fixed and stored for later analysis. - [Read Fluorescent Staining of Cell Surface Antigens on Viable Cells Using Direct Immunofluorescence]

Category: Microinjection Protocols: Microinjection of DNA Protocols

This protocol describes a method for constant-flow microinjection using the Pneumatic PicoPump (World Precision Instruments). This type of system is very simple and can be assembled on a relatively low budget. In this method, a constant flow of sample is delivered from the tip of the pipette, and the amount of sample injected into the cell is determined by how long the pipette remains in the cell. - [Read Gene Delivery by Direct Injection (Microinjection) Using a Controlled-Flow System Protocol]

Category: Microinjection Protocols: Microinjection of DNA Protocols

This protocol describes a method for pulsed-flow microinjection using the Eppendorf FemtoJet injector and Eppendorf InjectMan; this is the most common type of pulsed-flow microinjection system currently being used. The advantage of this type of system over a controlled-flow system is that much more control is available over the injection parameters, reducing variability in injections. In addition, the system allows a diagonal insertion of the needle into the cell. - [Read Gene Delivery by Direct Injection (Microinjection) Using a Pulsed-Flow System Protocol]

Category: DNA Microarray Protocols

Preparation of Fluorescent DNA Probe from HUMAN mRNA or Total RNA using Direct Incorporation Washing and Scanning Arrays. Brown Lab. - [Read Human DNA Microarray Hybridization]