Protocol for configuration, column construction, and column packing for a capillary liquid chromatography system. Protocol describes a procedure for adapting conventional HPLC systems to provide accurate low-flow rates (0.4-4 µl/min) and gradients required to operate slurry-packed capillary columns. A key component of this system is a commercial axial-beam longitudinal flow cell that can be fitted to several commercial UV detectors. - [Read Configuration Column Construction Column Packing for Capillary Liquid Chromatography]
Method of choice when large amounts of mammalian DNA are required, for example, for Southern blotting (Rapid Isolation of Mammalian DNA, Rapid Isolation of Yeast DNA, Southern Blotting: Capillary Transfer of DNA to Membranes) or for construction of genomic libraries in bacteriophage {lambda} vectors. Approximately 200 µg of mammalian DNA, 100-150 kb in length, is obtained from 5 x 107 cultured aneuploid mammalian cells (e.g., HeLa cells). - [Read Isolation of High-molecular-weight DNA from Mammalian Cells Using Proteinase K and Phenol Protocol]
Protocol describes a procedure for adapting conventional HPLC systems to provide accurate low-flow rates (0.4-4 µl/min) and gradients required to operate slurry-packed capillary columns. A key component of this system is a commercial axial-beam longitudinal flow cell that can be fitted to a number of commercial UV detectors. - [Read Packing Capillary Columns for RP-HPLC Protocol]
Protocol describes the first stages of Southern blotting: digestion of genomic DNA with one or more restriction enzymes, separation of the resulting fragments by electrophoresis through an agarose gel, and transfer of the denatured fragments to a membrane by downward capillary transfer. - [Read Southern Blotting: Capillary Transfer of DNA to Membranes Protocol]
Protocol describes the transfer of RNA from agarose gels to neutral or positively charged nylon membranes, using upward capillary flow of neutral or alkaline buffers. RNA becomes covalently fixed to positively charged nylon membranes during transfer in alkaline buffers. However, treatment by UV irradiation or heating is required to fix RNA to neutral membranes. - [Read Transfer and Fixation of Denatured RNA to Membranes Protocol]