incubated Protocols

Category: Reporter Gene Assays: B-Galactosidase Assay Protocols

The assay for ß-galactosidase relies on the ability of the enzyme to catalyze the hydrolysis of ONPG (o-nitrophenyl-ß-D- galactopyranoside) to free o-nitrophenol, which absorbs light at 420 nm. In this protocol, extracts of cells transfected with a ß-galactosidase reporter plasmid are incubated with ONPG. - [Read Assay for ß-galactosidase in Extracts of Mammalian Cells]

Category: Yeast Protocols: Yeast Genetics Protocols: B-Galactosidase Assay Protocols

In this protocol, extracts of cells transfected with a ß-galactosidase reporter plasmid are incubated with ONPG. - [Read Assay for ß-galactosidase in Extracts of Mammalian Cells Protocol]

Category: Bacterial Cell Culture Protocols: Bacterial Stocks Protocols

4 strains of E. coli are used in these studies: JM101 for M13 infection and isolation, XL1BMRF'for M13 or pUC-based DNA transformation, and ED8767 for cosmid DNA transformation. To maintain their respective F' episomes necessary for M13 viral infection, JM101 is streaked onto a M9 minimal media plate and XL1BMRF' is streaked onto an LB plate containing tetracycline. ED8767 is streaked onto an LB plate. These plates are incubated at 37degC overnight. For each strain, 3 ml. of appropriate liquid.. - [Read Bacterial Cell Maintenance Protocol]

Category: DNA Protein Interactions Protocols: Dnase Footprinting Assay Protocols

Protocol describes how isolated nuclei are incubated with varying amounts of Dnase I. Genomic DNA is then isolated from the nuclei and digested with a restriction enzyme, analyzed by gel electrophoresis, and probed by Southern hybridization. - [Read Mapping Dnase-I-hypersensitive Sites Protocol]

Category: Reporter Gene Assays: Cat Assays Protocols

In this protocol, extracts prepared from cells transfected with a chloramphenicol acetyltransferase (CAT) reporter plasmid are incubated with radiolabeled chloramphenicol. The acetylated products generated by the action of CAT are separated from the unmodified drug by thin-layer chromatography and quantitated by scraping the spots from the thin-layer plates and counting them by scintillation spectroscopy. - [Read Measurement of CAT in Extracts of Mammalian Cells Using Thin-layer Chromatography]

Category: Cell Biology and Cell Culture: In Vitro Reconstitution Protocols

In this protocol nuclei isolated from cells expressing the gene of interest are incubated with radiolabeled UTP which is incorporated into nascent RNA transcripts by RNA polymerase molecules that were actively transcribing at the time the cells were harvested. Because very little denovo initiation of RNA synthesis occurs in isolated nuclei, transcription of the target gene can be measured by hybridizing the radiolabeled RNA to an excess of the target gene immobilized on a nitrocellulose or nylon - [Read Protocol for Transcriptional Run- On Assays]

Category: Bacterial Protocols

Bacterial colonies growing on agar plates are transferred en masse to nitrocellulose filters. The spatial arrangement of colonies on the plates is preserved on the filters. After transfer, the filters are processed for hybridization to an appropriate radiolabeled probe while the original (master) plate is incubated for a few hours to allow the bacterial colonies to regrow in their original positions. - [Read Screening Bacterial Colonies by Hybridization: Intermediate Numbers Protocol]