including Protocols

Category: PCR Protocols

The MagneSil system can selectively isolate PCR products that are more than 150-bp long from primers and primer -dimers.  The technology can be used with a number of robotic workstations, including Beckman Coulter’s Biomek 2000 and FX Laboratory Automation Workstations.  The procedure can also be carried out manually.  Typical recovery is more than 80% for a 1-kb product with negligible carryover of primers or nucleotides. - [Read A Magnetic Particle-Based Method for Purifying PCR Products from Solution Protocol]

Category: Cytogenetics Protocols: Spectral Karyotyping SKY Protocols

SKY has been applied to various tumor groups including hematological malignancies, sarcomas, carcinomas and brain tumors, with the intent of identifying specific chromosomal abnormalities that may provide insight to the genes involved in the disease process as well as identifying recurrent cytogenetic markers for clinical diagnosis and prognostic assessment. - [Read Applications of SKY in Cancer Cytogenetics]

Category: Chromatography Protocols: Column Chromatography Protocols

Excellent PDF on Column Chromatography including detailed protocol and discussion. Univ. Tulane. - [Read Column Chromatography Protocol PDF]

Category: Yeast Cell Culture Protocols: Yeast Media, Solutions and Stocks Protocols

Complex yeast media protocols. Including: YPAD Medium or Also Called YPD plus Adenine; Synthetic Complete Drop Out (SC drop-out)Medium; Synthetic Complete drop-out Medium Mix (SC drop-out). - [Read Complex Yeast Media Protocols]

Category: PCR Protocols

Protocol for dealing with carryover contamination in PCR- enzymatic strategy. Repeated use of PCR and manipulation of its products cause aerosols that can contaminate neighboring samples and work areas.  Such "carryover contamination" can be prevented by including dUTP in place of dTTP for all amplification reactions. - [Read Dealing with Carryover Contamination in PCR: An Enzymatic Strategy Protocol]

Category: Proteomic Protocols: 2-D Gel Electrophoresis

Excellent Detailed Protocol for 2-D Gel Electrophoresis including staining, gel running, and equipment. Purdue PSAL. - [Read Detailed 2D Gel Electrophoresis]

Category: Cytogenetics Protocols: In Situ Hybridization Tissue Sections Protocols

Protocol for detection of even-skipped transcripts in drosophila embryos with PCR/DIG-labeled DNA probes. This protocol has been used to detect the transcript distribution of a number of genes by in situ hybridization, including evenskipped and seven-up, in whole mount Drosophila embryos, and engrailed Antennapedia in whole mount grasshopper embryos. Includes: Probe labeling; Evaluation of labeling reaction; Preparation of embryos, hybridization and detection.
 - [Read Detection of Even-Skipped Transcripts in Drosophila Embryos with PCR/DIG-Labeled DNA Probes Protocol]

Category: DNA Protocols: DNA Extraction Protocols: Agarose Gel DNA Extraction Protocols

DNA Extraction from Agarose Gels Protocol. The page includes cutting out the DNA band from the gel, and describes three methods including 1) Spin-columns (Nucleic acid purification columns), 2) using Dialysis tubing (semi-permeable membrane, Visking tubing), and the 3) Paper strip method.Matt Lewis, Department of Pathology, University of Liverpool. - [Read DNA Extraction from Agarose Gels Protocol]

Category: PCR Protocols: PCR Optimization

Method for amplifying DNA enzymatically by the polymerase chain reaction (PCR), including procedures to quickly determine conditions for successful amplification of the sequence and primer sets of interest, and to optimize for specificity, sensitivity, and yield.  The first step of PCR simply entails mixing template DNA, two appropriate oligonucleotide primers, Taq or other thermostable DNA polymerases, deoxyribonucleoside triphosphates (dNTPs), and a buffer. - [Read Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization Protocol]

Category: DNA Protocols: DNA Extraction Protocols

DNA isolation from various fungal species including: Cochliobolus, Aternaria, and Fusarium. Key steps: (1) the use of young lyophilized mycelial mats - yield less contaminating carbohydrates; (2) proteinase K in the extraction buffer to destroy DNases (f - [Read Fungal DNA Isolation Method]

Category: Viral Culture and Isolation Protocols: Viral Culture Protocols

Most manipulations with M13, including preparations of viral stocks and isolation of single- and double-stranded DNAs, begin with small-scale liquid cultures that are infected with an M13 plaque, picked from an agar plate. - [Read Growing Bacteriophage M13 in Liquid Culture Protocol]

Category: Laboratory Model Organisms Protocols

Protocol describes the growth and concentration of the alga Chlorogonium elongatum as a food source for culturing freshwater hypotrichs. Most freshwater hypotrichs (including Oxytricha nova, O. fallax, and O. trifallax; Euplotes aediculatus and E. eurystomous; and Stylonychia lemnae) can be grown to high density with Chlorogonium as the food organism. A similar regimen can be used to prepare other food sources such as Tetrahymena or bacteria (e.g., Aerobacter aerogenes). - [Read Growth and Concentration of Chlorogonium for Culturing Freshwater Hypotrichs Protocol]

Category: Plant Biology Protocols: Plant Genetics Protocols

To accurately predict the activity of a transgene it is critical to understand its location and dynamics in the 3-D interphase nucleus. Developed in situ methods to visualize transgenes (including single copy genes) & their transcripts during interphase from different tissues & plant species. These techniques reduce the time necessary for characterization of transgene integration by eliminating the need for time-consuming segregation analysis and extend characterization to the interphase nucleus - [Read In Situ Methods to Localize Transgenes and Transcripts in Interphase Nuclei]

Category: Cytogenetics Protocols: In Situ Hybridization Protocols

In situ methods to visualize transgenes (including single copy genes) and their transcripts during interphase from different tissues and plant species. These techniques reduce the time necessary for characterization of transgene integration by eliminating the need for time-consuming segregation analysis, and extend characterization to the interphase nucleus, thus increasing the likelihood of accurate prediction of transgene activity. - [Read In Situ Methods to Localize Transgenes and Transcripts in Interphase Nuclei]

Category: Immunology: T Cell Protocols

Peyer’s Patch, and Lamina Propria Cells lymphocyte populations should be analyzed when studying the immunological status of the intestine, for example in oral immunization or in intestinal disease (including infectious disease and tumors). This protocol details techniques for isolation of IEL, PP cells, and LP cells from the small intestine of the mouse. - [Read Isolation of Mouse Small Intestinal Intraepithelial Lymphocytes Protocol]

Category: Molecular Model Organisms: Mouse Animal Protocols: Mouse Genotyping Protocols

Contains the NHLBI Division of Intramural Research, including a list of labs, and the Office of Technology Transfer and Development. - [Read Laboratory of Developmental Biology, Division of Intramural Research, NHLBI, NIH]

Category: C. elegans Protocols

At low magnification, dissection microscopes are useful, whereas at higher magnification and resolution, a wide range of optical methods can be used, including differential interference contrast (DIC) optics, phase contrast, fluorescence, polarized light, confocal, and dark field. - [Read Live Imaging of Caenorhabditis elegans: Observation of Nematodes and Data Collection Protocol]

Category: Protein Protocols: Lowry Protein Assay

Lowry Protein Assay Protocol. Yu-li Wang Lab, Univ. Massachusetts Medical School. Detailed protocol including materials. - [Read Lowry Protein Assay Protocol]

Category: Bacterial Cell Culture Protocols: Bacterial Stocks Protocols

Plasmid (pUC series) containing genomic DNA fragments are maintained in E. coli strain DH5aTM. The E. coli cultures are routinely cultured at 37 C on Luria-Bertani (LB) agar on or in LB broth containing Ampicillin (30 µg/ml) or Carbenicillin (50 µg/ml broth, 100 µg/ml agar). E. coli strains are usually preserved in stab agar or glycerol for mid-term storage and lyophilized for long-term storage. - [Read Maintenance of Probes in Bacteria Including Escherichia coli Protocol]

Category: Cell Biology and Cell Culture: Cell Adhesion Protocols

Many proteins and molecules promote cell adhesion including several cell surface carbohydrate binding proteins. Cell adhesion measurements on 96-well microtiter plate format are difficult due to the shear forces generated by washing the wells. The protocol here introduces the use of a liquid-filled wash chamber that separates unbound cells by gravity. This eliminates uncontrolled shear forces and passage of adherent cells through a liquid/air interface. John L. Magnani~GlycoTech Corporation. - [Read Measurement of Cell Adhesion Under Static Conditions]

Category: Microscopy Protocols: Live-Cell Imaging Protocols

Several common drugs, their targets, and protocols are described for studying organelle distribution and trafficking. The drugs are readily available from general suppliers, including Sigma, Roche, and Calbiochem. - [Read Membrane Trafficking and Organelle Reagents]

Category: Mass Spectrometry Protocols

Methanol-Chloroform Precipitation of Proteins. A procedure for precipitating proteins from solution, including detergent solutions, is from Wessel and Flügge (1984). Univ. Virginia. - [Read Methanol-Chloroform Precipitation of Proteins]

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

Exponentially growing cells are asynchronous with respect to the cell cycle stage. Detection of cell cycle-related events is improved by enriching the culture for cells at the stage during which the particular event occurs. Methods for synchronizing cells are provided here, including those based on morphological features of the cell. - [Read Methods for Synchronizing Cells at Specific Stages of the Cell Cycle]

Category: Genetics and Genomics: Genotyping Protocols: PCR Genotyping

Protocol for PCR genotyping from tail DNA. This protocol works well for a variety of genes and primer pairs including Tg and KO alleles. Oligonucleotide melting temperatures between 60° and 65° seem to work well. - [Read PCR Genotyping from Tail DNA Protocol]

Category: Immunology: T Cell Protocols

Provides methods for the derivation of specific types of T cell clones: preparation and maintenance of alloreactive murine helper T (TH) lymphocyte and cytotoxic T lymphocyte (CTL) clones using the limiting dilution technique and derivation of TH clones reactive with soluble protein antigens including a method for the selection of either TH1 or TH2 lymphocyte subsets. - [Read Production of T Cell Clones Protocol]