generally Protocols

Category: Nucleic Acid Purification Protocols

Protocol for a single-step method for the simultaneous preparation of DNA, RNA, and protein from cells and tissues. The yield of total RNA depends on the tissue or cell source, but it is generally in the range of 4-7 µg/mg starting tissue or 5-10 µg/106 cells.  IMPORTANT: Prepare all reagents used in this protocol with Diethyl pyrocarbonate (DEPC)-treated H2O. - [Read A Single-step Method for the Simultaneous Preparation of DNA, RNA, and Protein from Cells and Tissue]

Category: DNA Protocols: Genomic DNA Library Protocols

This stage summarizes how to construct a cDNA library in a mammalian vector, using commercial kits. These kits are available from several manufacturers and generally include all the required reagents. - [Read Construction of cDNA Libraries in Eukaryotic Expression Vectors for Construction and Screening]

Category: Bacterial Cell Culture Protocols: Bacterial Cell Culture Media Protocols

This protocol has been used successfully to 15N or 13C/15N label our proteins using our pET1120/BL21(DE3) expression system: Preparing M9 minimal media begins with preparing a 5x stock solution of M9 salts. Generally, M9 salts contain a nitrogen source in the form of NH4Cl. Since we want to add a labeled nitrogen source, our 5x salts are prepared minus NH4Cl. Standard 5 X M9 Minimal Media salts minus nitrogen source For 1L 5xM9 salts: - [Read Expression Protocol in M9 Minimal Media via T7 Promoter]

Category: Cell Organelle Protocols: Subcellular Fractionation Protocols: Mitochondria Isolation Protocols

Generally in iodixanol gradients the density of organelles decreases in the series: peroxisomes, mitochondria, lysosomes, ER, Golgi, although in Dictyostelium discoideum, the lysosomes are denser than the mitochondria. Iodixanol gradients can usually provide satisfactory resolution of all these membrane particles although it may be necessary to modulate either the gradient or centrifugation parameters in order to optimize a particular separation. - [Read Fractionation of Mitochondria, Lysosomes, Peroxisomes, ER and Golgi in Pre-formed Iodixanol Gradient]

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols: DNA Isolation from Mammalian Cells Protocols

Procedure is used to prepare DNA simultaneously from many different types of samples or tissues.  Although the DNA is generally too small (approx.  80 kb) for efficient construction of genomic DNA libraries, it gives excellent results in Southern hybridizations and PCRs.  Cultured aneuploid mammalian cells (2 x 107, e.g., HeLa cells) yield 100 µg of DNA in a volume of 1 ml. - [Read Isolation of DNA from Mammalian Cells by Spooling 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: Histology Protocols: Tissue Sample Sectioning

Generally Required Materials and Equipment for Tissue Sectioning. Sectioning and Slide Preparation Procedure onto Metal-Framed PEN Membrane Slides. Arcturus. - [Read Optimized Protocol for Mounting Tissue Sections PDF]

Category: Viral Manipulation Protocols: M13 Phage Manipulation Protocols

Bacteriophage M13 single-stranded DNA is prepared from virus particles secreted by infected cells into the surrounding medium. The filamentous particles are concentrated by precipitation from a high-ionic-strength buffer with polyethylene glycol. Subsequent extraction with phenol releases the single-stranded DNA, which is then collected by precipitation with ethanol. This protocol is generally used to prepare single-stranded DNA from a small number of M13 isolates. - [Read Preparation of Single-stranded Bacteriophage M13 DNA Protocol]

Category: Cell Biology and Cell Culture: Cell Cycle Protocols

This method works well to assess cell cycle distribution of whole cell populations. This method can also be used to assess the cell cycle distribution of GFP transfected cells however, the EtOH step is generally not sufficient to keep GFP in the cell. - [Read Preparing Cells for PI/FACS (cell cycle) Analysis Protocol]

Category: Nucleic Acid Purification Protocols: RNA Isolation Protocols: RNA Isolation fromTissues or Cultured Cells Protocols

Single-step technique, cells are homogenized in guanidnium thiocyanate and the RNA is purified from the lysate by extraction with phenol:chloroform at reduced pH.  Many samples can be processed simultaneously and speedily.  The yield of total RNA depends on the tissue or cell source and is generally in the range of 4-7 µg/ml starting tissue or 5-10 µg/106 cells.  IMPORTANT: Prepare all reagents used in this protocol with Diethyl pyrocarbonate (DEPC)-treated H2O. - [Read Purification of RNA from Cells and Tissues by Acid Phenol-Guanidinium Thiocyanate-Chloroform Extract]

Category: Cytoskeleton Protocols: Microtubules Protocols

Recycle tubulin fractions stored at -80¡C after the PC column and store the recycled tubulin in small aliquots for day-to-day use. Generally store recycled tubulin in Injection Buffer (IB) without free GTP. This is done because depolymerization appears to be much better in IB, IB is ideal for microinjections/adding tubulin to extracts, and the absence of free GTP makes polymerization with GMPCPP, a very useful GTP analog that has ~5-10X lower affinity than GTP for tubulin. - [Read Recycling Tubulin Protocol]

Category: Chromatography Protocols: Dye Ligand Affinity Chromatography Protocols

Protocol for screening immobilized dyes for their ability to bind a target protein. The selection of a dye adsorbent for the purification of the target protein is an empirical undertaking and is generally achieved by a trial-and-error approach. - [Read Screening Immobilized Dyes for their Ability to Bind a Target Protein Protocol]

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: Viral Manipulation Protocols: Phage Protocols

Protocol describes methods for recovery and purification of recombinant clones of bacteriophage P1 or PAC DNAs from bacteria. Because of their large size, these DNAs are sensitive to shearing forces and must be handled carefully. This protocol generally yields P1 DNA that works well as a substrate or template in enzymatic reactions. - [Read Working with Bacteriophage P1 and Its Cloning Systems Protocol]