double Protocols

Category: Cloning Protocols

Adaptors are short double-stranded synthetic oligonucleotides that carry an internal restriction endonuclease recognition site and single-stranded tails at one or both ends. Adaptors are used to exchange restriction sites at the termini of linear DNA molecules. They may be purchased in phosphorylated and unphosphorylated forms. - [Read Attaching Adaptors to Protruding Termini Protocol]

Category: DNA Protocols: cDNA Library Protocols

This stage achieves four goals: polishing the ends of double-stranded DNA, ligation of synthetic linkers or adaptors, digestion of the attached linkers to create cohesive termini, and preparing the cDNA for cloning. - [Read Attachment of Linkers or Adaptors for Construction of cDNA Libraries]

Category: Cell Organelle Protocols: Nucleus Protocols

The key step is the lysis which solubilizes centrosomes away from nuclei by very low ionic strength lysis after treatment of cells with nocodazole and cytochalasin B. The released centrosomes are then centrifuged onto a Ficoll cushion (to avoid pelleting) and the interface between the lysate and the Ficoll is collected and the centrosomes are concentrated on a sucrose gradient. Fractions are assayed by spindown and double IF with 5051 serum and anti-tubulin and the pooled fractions are frozen... - [Read CHO Centrosome Prep 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: RNAi Technology Protocols: RNAi Mouse Embryos and Oocytes Protocols

Protocol describes a method to collect early embryos from 6-week-old mice.  Subsequently, the isolated embryos can be injected with double-stranded RNA to induce knockdown of a gene of interest. - [Read Collection of Early Mouse Embryos for RNAi Protocol]

Category: RNAi Technology Protocols: RNAi Mouse Embryos and Oocytes Protocols

Protocol describes a method to collect oocytes from 6-week-old mice.  Subsequently, the isolated oocytes can be injected with double-stranded RNA to induce knockdown of a gene of interest. - [Read Collection of Mouse Oocytes for RNAi Protocol]

Category: Microbiology: Bacterial Competent Cells Preparation E.Coli

Bacterial E.Coli competent cell preparation. Calcium Chloride Protocol, Preparation of calcium chloride competent cells for frozen storage, Electroporation Protocol, Electroporation Protocol for transformations using double-stranded plasmids, Reference: - [Read Competent cell preparation]

Category: DNA Protocols: cDNA Library Protocols

Here, the DNA-RNA hybrids synthesized in Stage 1 are converted into full-length double-stranded cDNAs. The primers for synthesis of second-strand cDNA are created by RNase H, which introduces nicks into the RNA moiety of the cDNA-mRNA hybrids. E. coli DNA polymerase I extends the newly created 3'-hydroxyl termini, using the first-strand cDNA as a template. - [Read Construction of cDNA Libraries Protocol]

Category: DNA Protocols: cDNA Library Protocols

The goal of this stage is to introduce methyl groups that will modify and protect naturally occurring EcoRI sites in the double-stranded cDNA. - [Read Construction of cDNA Libraries Protocol.]

Category: DNA Protocols: Cosmid Library Protocols

Protocol describes how to construct a library of 35-45-kb fragments of genomic DNA in the double cos site cosmid vector, SuperCos-1. The steps include: Linearization and dephosphorylation of SuperCos-1 DNA; Partial digestion of high-molecular-weight DNA with MboI; Dephosphorylation of high-molecular-weight genomic DNA; Ligation of cosmid arms to genomic DNA: Packaging and plating recombinants; Isolation and analysis of recombinant cosmids: Validation of the library. - [Read Construction of Genomic DNA Libraries in Cosmid Vectors Protocol]

Category: RNAi Technology Protocols: RNAi Drosophila Protocols

Protocol describes how subcellular-sized particles are accelerated to high velocity to carry double-stranded RNA (dsRNA) into Drosophila embryos.  The major advantage of this procedure over microinjection (Microinjection of dsRNA into Drosophila Embryos) is that particle bombardment is easier and faster to perform.  In addition, the mechanical trauma received is far less than by microinjection, allowing better survival of embryos and fewer phenotypic artifacts. - [Read Delivery of dsRNA into Drosophila Embryos by Gene Gun Protocol]

Category: Nucleic Acid Electrophoresis Protocols: DNA Electrophoresis Protocols: DNA Electrophoresis in Agarose Gels Protocols

The most convenient and commonly used method to visualize DNA in agarose gels is staining with the fluorescent dye ethidium bromide.  Ethidium bromide can be used to detect both singleand double-stranded nucleic acids (both DNA and RNA).  However, the affinity of the dye for single-stranded nucleic acid is relatively low and the fluorescent yield is comparatively poor. - [Read Detection of DNA in Agarose Gels Protocol]

Category: Histology Protocols: Histology Staining Procedures

Protocol for double AP staining. - [Read Double AP Staining Protocol]

Category: C. elegans Protocols: C. elegans Staining Protocols

Protocol for double dye filling (using DiO and di-4 ANEPPS). - [Read Double Dye Filling (using DiO and di-4 ANEPPS) Protocol]

Category: Molecular Imaging: Immunofluorescence Microscopy

Double immunofluorescence staining for BCL-6. unfixed or acetone-fixed specimens. dewaxed, antigen retrieved slides - [Read Double immunofluorescence staining for BCL-6]

Category: Immunology: Immunofluorescence Protocols: Immunofluorescence Staining Protocols

Protocol for double immunofluorescence staining for BCL-6. Any type of tissue is suitable for this technique, as long as the antigenicity for your antigen(s) is preserved. Includes: unfixed or acetone-fixed specimens; dewaxed, antigen retrieved slides. - [Read Double Immunofluorescence Staining for BCL-6 Protocol]

Category: Drosophila Protocols: Drosophila Genetics Protocols

Protocol for Drosophila non-fluorescent protein and RNA double-labeling. - [Read Drosophila Non-fluorescent Protein and RNA Double-Labeling Protocol]

Category: Nucleic Acid Modification Protocols: Nucleic Acid Radiolabeling Protocols

Protocol for end labeling protruding 3' termini of double-stranded DNA with [{alpha}-32P]Cordycepin 5'-Triphosphate or [{alpha}-32P]dideoxyATP. - [Read End Labeling Protruding 3' Termini of Double-stranded DNA Protocol]

Category: Drosophila Protocols: Drosophila Genetics Protocols

FISH protocols for Drosophila. Includes: RNA Probe Preparation; Embryo Collection and Fixation; Single FISH on Drosophila embryos; Post-Fixation, Hybridization and Post-Hybridization Washes; Development of FISH Signal; Storage, Mounting and Viewing of Samples; Double FISH on Drosophila Embryos; RNA-Protein Double Labeling; FISH on Dissected Tissues. - [Read FISH Protocols for Drosophila]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols

FIXATION and DNA Staining for Cell Cycle Analysis Protocol. This method of DNA staining utilizes ethanol to fix the cells and permeabilize the membrane, which allows the dye (Propidium Iodide) to enter the cells.  Propidium Iodide (PI) is a DNA-binding fluorochrome that intercalates in the double-helix.  Ribonuclease-A is used to eliminate the staining of double-stranded RNA. - [Read FIXATION and DNA Staining for Cell Cycle Analysis]

Category: Nucleic Acid Detection Protocols

Protocol describes the quantitation of DNA using Hoechst 33258, a fluorescent dye that binds to double-stranded DNA.  Fluorometry is simple and more sensitive than spectrophotometry, and allows the detection of nanogram quantities of DNA.  The assay can only be used to measure the concentration of DNAs whose sizes exceed ~1 kb, as Hoechst 33258 binds poorly to smaller DNA fragments. - [Read Fluorometric Quantitation of DNA Using Hoechst 33258 Protocol]

Category: Fungi Protocols: Aspergillus Protocols

Compendium of protocols for using Aspergillus nidulans in genetic, molecular, and cell biological investigations, originally written for members of my research group. It also summarizes our common growth media and nutritional supplements, many of which originally appeared elsewhere but now are difficult to locate. Includes: Growth and storage of Aspergillus nidulans conidia; Nutritional supplements for our common auxotrophies; Double mutants; Mitotic mapping - assigning genes to chromosomes; etc - [Read Fundamentals of Growth, Storage, Genetics and Microscopy of Aspergillus nidulans Protocols]

Category: Cell Biology and Cell Culture: Cell Cycle Protocols: S Phase Protocols

This protocol provides a method for synchronizing cells at the G1/S border using a double treatment of thymidine, which, in excess, is an inhibitor of DNA synthesis. Cells are treated once with excess thymidine to accumulate the majority of them at G1/S; however, some cells will have stopped growth within the S phase. - [Read G1/S Phase Synchronization using Double Thymidine Synchronization Protocol]

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

This protocol provides a method for synchronizing cells at the G1/S border using a double treatment of thymidine, which, in excess, is an inhibitor of DNA synthesis. Cells are treated once with excess thymidine to accumulate the majority of them at G1/S; however, some cells will have stopped growth within the S phase. - [Read G1/S Phase Synchronization using Double Thymidine Synchronization Protocol]

Category: Cell Biology and Cell Culture: Cell Cycle Protocols: G Phase Protocols

This protocol provides a method for synchronizing cells at the G1/S border using a double treatment of thymidine, which, in excess, is an inhibitor of DNA synthesis. Cells are treated once with excess thymidine to accumulate the majority of them at G1/S; however, some cells will have stopped growth within the S phase. - [Read G1/S Phase Synchronization using Double Thymidine Synchronization Protocols]