increase Protocols

Category: PCR Protocols: PCR Cloning Protocols

Protocol for blunt-end cloning of PCR products. Incubation of a blunt-end ligation reaction in the presence of an excess amount of an appropriate restriction enzyme can dramatically increase the yield of recombinant plasmids.  The role of the restriction enzyme is to cleave circular and linear concatemers at restriction sites that are re-formed when linear, blunt-ended plasmid molecules ligate to themselves.  I - [Read Blunt-end Cloning of PCR Products Protocol]

Category: Microinjection Protocols: Microinjection Equipment

This protocol describes an easy method for calibrating micropipette tips that have been pulled in the laboratory. It is essential to estimate the internal diameter of the pulled micropipette tip when adjusting parameters for a new puller or new type of glass tubing. A tip diameter of ~0.3 µm is optimal for the microinjection of mammalian cells in culture (e.g., CHO, PtK1, and COS-7). A 10% increase in diameter increases the delivery rate by more than 30% and can cause cell damage. - [Read Calibration of Micropipette Tips Protocols]

Category: Cell Organelle Protocols: Subcellular Fractionation Protocols

A number of density gradient strategies have been developed for the fractionation of human erythrocytes according to their age. As the cells age, so their density tends to increase; reticulocytes therefore tend to have the lowest densities. Reticulocytes have frequently been partially purified on discontinuous gradients of arabinogalactan; the actual density range being quite varied, from quite broad ones. - [Read Fractionation of Human Erythrocytes (Normal or Sickle) and Reticulocytes in Discontinuous Iodixanol]

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: Chromatography Protocols: Dye Ligand Affinity Chromatography Protocols

Protocol for the optimization of absorption condition for dye-ligand affinity chromotography. Generally, low pH and low ionic strength, absence of phosphate ions, and the presence of divalent metals ions increase the binding of proteins to immobilized triazine dyes. - [Read Optimization of Adsorption Conditions for Dye-Ligand Affinity Chromatography Protocol]

Category: Cell Biology and Cell Culture: Cell Cycle Protocols: Mitosis Protocols

Protocol describes a method for synchronizing monolayer cells in mitosis using selective detachment from their substrate. During mitosis, cells become spherical, causing them to become more loosely attached to their substrate. The "rounded up" cells are selectively detached by tapping the culture flask, resulting in a population in which as many as 90-98% of the cells are in mitosis. The drug nocodazole is used to increase the percentage of cells undergoing mitosis before detachment is performed - [Read Synchronization of Mammalian Cell Cultures in Mitosis Using Selective Detachment Protocol]

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

This protocol describes a method for synchronizing monolayer cells in mitosis using selective detachment from their substrate. During mitosis, cells become more spherical, causing them to become more loosely attached to their substrate. The "rounded up" cells are selectively detached by tapping the culture flask, resulting in a population in which as many as 90-98% of the cells are in mitosis. The drug nocodazole is used to increase the percentage of cells undergoing mitosis before detachment.. - [Read Synchronization of Mammalian Cell Cultures in Mitosis Using Selective Detachment Protocol]

Category: Western Blot Protocols: Antigen Detection Methods Protocols

Use of the chemiluminescence-producing alkaline phosphatase substrate 3-(4-methoxyspiro[1,2-dioxetane-3,2'-tricyclo-[3.3.1.1(3,7)]decan]-4-yl)phenyl phosphate (AMPPD, also known as adamantyl-1,2-dioxetane phosphate), or its dioxetane relatives provides a substantial increase in sensitivity over colorimetric substrates and radiochemical methods currently used for the detection of antigen-antibody complexes immobilized on nylon or PVDF membranes. - [Read Western Analysis Using the Chemiluminescent Alkaline Phosphatase Substrate CSPD Protocol]

Category: Plant Biology Protocols: Plant DNA RNA Isolation Protocols

During development many plant cells undergo endoreduplication, whereby ploidy increases to a multiple of the normal 2C content. For eg., trichome development is accompanied by an increase in ploidy to 32C, indicating that trichome cells undergo four rounds of endoreduplication. Protocol describes DNA levels, and hence developmental progress in the corresponding cells, are measured by staining the DNA with a fluorescent marker and then quantifying the fluorescence of individual nuclei. - [Read Whole-Mount DAPI Staining and Measurement of DNA Content in Plant Cells]