adapted Protocols

Category: Cell Culture Protocols: Insect Cell Culture Protocols

This protocol is designed to adapt High Five™ (BTI-Tn-4) cells to serum-free suspension culture in HyQ SFX-Insect in 5 passages. The entire adaptation process takes approximately two weeks and the resulting adapted cells have doubling times of 16 and 20 hours and minimal clumping in suspension culture. - [Read Adaptation of High Five™ Cells to HyQ© SFX Insect Medium]

Category: Epigenetics and Methylation Protocols

Bisulfite Treatment of DNA Anderson Lab. Adapted from Frommer et.al. - [Read Bisulfite Treatment of DNA Anderson Lab]

Category: Epigenetics and Methylation Protocols: Bisulfite Treatment Methylation Assay Protocol

Adapted from Frommer et.al. Good protocol for bisulfite treatment of DNA. Includes tips on Methylation PCR for CpG methylation analysis. University of Texas M. D. Anderson Cancer Center - [Read Bisulfite Treatment of DNA for Methylation Analysis]

Category: Transfection Protocols: Calcium Phosphate Transfection Protocols

Calcium phosphate forms an insoluble precipitate with DNA, which attaches to the cell surface and is taken into the cells by endocytosis. The protocol is easily adapted for use with other types of cells, both adherent and nonadherent. This protocol is a modified version of a method published by Jordan et al. (1996) who rigorously optimized calcium-phosphate-based transfection methods for Chinese hamster ovary cells and the 293 line of human embryonic kidney cells. - [Read Calcium-phosphate-mediated Transfection of Eukaryotic Cells with Plasmid DNAs]

Category: Oligonucleotide Protocols: Oligonucleotide Purification Protocols

Concentration of DNA by Ethanol Precipitation Protocol. Adapted from Bruce A. Roe, Department of Chemistry and Biochemistry, The University of Oklahoma, Norman, Oklahoma. Usually 2.5 - 3 volumes of  ethanol and/or acetate solution is added to the DNA in a microcentrifuge tube. This is then put into an ice-water bath for at least 10 minutes. The precipitation is performed by incubation at -20C overnight. - [Read Concentration of Oligo DNA by Ethanol Precipitation Protocol]

Category: Cell Culture Protocols: Insect Cell Culture Protocols

Using this method, insect cells should be adapted to HyQ SFX-Insect in 4-8 passages. However, there are some cell lines and clones that are more sensitive to the physiochemical and nutritional changes, and in some cases it may take longer than 8 passages for the adaptation. - [Read Direct Adaptation of Insect Cells to HyQ© SFX-Insect™ Medium]

Category: Genetics and Genomics: Genotyping Protocols: Mutation Detection Protocols

The goal of this method is to identify transcriptionally active genes in cloned segments of genomic DNA. The protocol uses hybridization and affinity purification to recover biotin-labeled cDNAs that bind to a 500-kb segment of human DNA cloned in a BAC vector. However, the method can be easily adapted to other clones of genomic DNAs cloned in high-capacity vectors. - [Read Direct Selection of cDNAs with Large Genomic DNA Clones Protocol]

Category: Protein Protocols: Protein Expression Protocols

Expression of F-tryptophan labelled protein. Protocol can be adapted for Fluoro-phenylalanine or Fluro-tyrosine labelling. Transformation of competent E. coli cells for expression with plasmid. Dr. Chen, Dept. of Biochem. & Mol. Biology, University College, London. - [Read Expression of F-tryptophan labelled protein]

Category: Viral Manipulation Protocols: Phage Lamda Manipulation Protocols

DNA is best isolated from bacteriophage lambda particles by digesting the viral coat proteins with a powerful protease such as proteinase K, followed by extraction with phenol:chloroform. This procedure is easily adapted for use with smaller-scale preparations of bacteriophage lambda. - [Read Extraction of Bacteriophage lambda DNA from Large-scale Cultures Using Proteinase K and SDS Protocol]

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

This protocol provides a method for the synchronization of a monolayer culture of CHO cells in G1 using isoleucine deprivation. Since CHO cells can also be adapted to grow in suspension culture, this procedure can be used to obtain larger quantities of cells. When isoleucine is replaced, the cells resume growth and begin to enter S phase ~4 hours later. This method arrests almost 100% of the CHO cells in G1, and upon reversal, leads to rapid recovery of cell growth and very high cell viability. - [Read G1 Synchronization of CHO Cells by Isoleucine Deprivation Protocol]

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

This protocol provides a method for the synchronization of a monolayer culture of CHO cells in G1 using isoleucine deprivation. Since CHO cells can also be adapted to grow in suspension culture, this procedure can be used to obtain larger quantities of cells. When isoleucine is replaced, the cells resume growth and begin to enter S phase ~4 hours later. This method arrests almost 100% of the CHO cells in G1, and upon reversal, leads to rapid recovery of cell growth and very high cell viability. - [Read G1 Synchronization of CHO Cells by Isoleucine Deprivation Protocol]

Category: Cell Culture Protocols

Glass is an excellent substrate for most tissue-culture-adapted cells and is compatible with all fixing and staining solutions. Glass coverslips in tissue-culture dishes or in 24-well multiwell plates are suitable carriers, as are multiwell slides. For high-resolution studies, choose glass coverslips of the highest available grade; #1 or #1.5 coverslips are the appropriate thickness. - [Read Growing Adherent Cells on Coverslips or Multiwell Slides Protocol]

Category: Immunohistochemistry Protocols: Cryosectioning Protocols

This protocol describes methods for the fixation, cryosectioning, and immunohistochemical detection of proteins in embryonic and adult zebrafish eyes. The methods described can easily be adapted for use on other zebrafish tissues. - [Read Immunohistochemistry on Cryosections from Embryonic and Adult Zebrafish Eyes Protocol]

Category: Laboratory Model Organisms Protocols

Protocol describes methods for the fixation, cryosectioning, and immunohistochemical detection of proteins in embryonic and adult zebrafish eyes. The methods described can easily be adapted for use on other zebrafish tissues. - [Read Immunohistochemistry on Cryosections from Embryonic and Adult Zebrafish Eyes Protocol]

Category: Immunohistochemistry Protocols

Describes two methods for using the immunoperoxidase reaction to localize antigens at the electron microscope level; one for adherent cultured cells and one for tissue sections. The reaction conditions are first optimized at the light microscope level and then adapted for EM level observation. These methods allow for reliable detection of antigens at the cell surface, within the cell, and especially in membrane bounded organelles. - [Read Immunoperoxidase Methods for Localization of Antigens in Cultured Cells and Tissues]

Category: Bacterial Protocols

Protocol was developed to isolate Wolbachia from adult Drosophila, but it can be adapted for other insects. In some insects leg removal prior to isolation facilitates hemolymph extrusion. - [Read Isolation of Live Bacteria from Adult Insects Protocol]

Category: Antibody Protocols: Antibody Labeling Protocols

This protocol describes a simple chemical oxidation method for labeling antibodies with iodine. Iodide-125 (supplied as NaI) is oxidized to form iodine-125 (I2), which attacks tyrosyl and histidyl side chains. The iodinated antibodies are easily detected and quantitated using gamma counters or film. They are used primarily in immunoassays, but other techniques can be adapted conveniently to the iodine detection method. - [Read Labeling Antibodies with Iodine Protocol]

Category: Yeast Protocols: Yeast Staining Protocols

Protocol describes our method for preparing cells for immunofluorescence, in which all incubations and washes are performed in microtiter dishes. The protocol can also easily be adapted for preparing cells for immunofluorescence in microfuge tubes. - [Read Large-Scale Immunocytology Protocol]

Category: Plant Biology Protocols: Plant DNA RNA Isolation Protocols

Protocol is based on the standard Trizol protocol for the purification of RNA from animal cells using Trizol (Purification of RNA from Animal Cells using Trizol). In this version, adapted for use with plant tissues, a high-salt isopropanol precipitation step has been added to precipitate RNA selectively, while maintaining polysaccharides and proteoglycans in solution. - [Read Preparation of RNA from Plant Tissue Using Trizol]

Category: Reporter Gene Assays: GFP Assay Protocols

Protocol specifically describes data acquisition for a particular variant of GFP (EGFP) or Oregon Green as a donor fluorophore, but it can be adapted for image acquisition of other chromophore. - [Read Probing Protein Interactions Using GFP and FRET]

Category: RNA Protocols: RT-PCR and cDNA synthesis

This procedure was first described by Bertrand et al to demonstrate that ribozymes could be enzymatically active in vivo. We adapted the method to show that certain oligodeoxynucleotides could direct the activity of endogenous ribonuclease H to cleave tar - [Read Reverse Ligation Mediated RT - PCR]

Category: Protein Protocols: Western Blot Protocol

Adapted from existing protocols by Vinh PhamLast modified: June 5, 2003. - [Read SDS-PAGE & Western Blotting Protocols]

Category: Protein Protocols: Protein Electrophoresis: SDS-PAGE Electrophoresis Protocols

SDS-PAGE Gels. Adapted from David Bowtell - [Read SDS-PAGE Gels]

Category: Cell Biology and Cell Culture: ES Stem Cell Biology and Culture

Subcloning Protocol ES Cells. Used an adapted form of the feeder-free protocols detailed in Xu et al. (Nature Biotechnology 19:971–974, 2001). NIH Stem Cell Unit - [Read Subcloning Protocol ES Cells]

Category: Protein Protocols: Protein Precipitation Procols

TCA Acetone precipitation protocol DOC. Matt Shipman. 2006. Adapted and modified from Gruehler et al., 2005. - [Read TCA Acetone precipitation protocol DOC]