hours Protocols

Category: Microscopy Protocols: Live-Cell Imaging Protocols

This protocol describes a sealed preparation that allows the continuous long-term observation of cultured mammalian cells on upright or inverted microscopes without environmental CO2 control. The preparation allows for optical conditions consistent with high-quality imaging and good cell viability for at least 100 hours. - [Read A Sealed Preparation for Long-Term Observations of Cultured Cells]

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: Cell Biology and Cell Culture: Cell Culture Techniques: Cell Line Preservation Freezing

Freezing gealthy cells (> 95% viable) the protocol obtains a culture 80-90% viable 24 hours after thawing and growing on test vial from step 9. Antisense Research Group - [Read Cell Freezing using Liquid Nitrogen N2]

Category: Xenopus Protocols: Xenopus Egg Extracts Protocols

Protocol for cytostatic factor extract preparation for spindle assembly. The quality of the eggs is essential for good CSF extracts. Always sacrifice quantity for quality when trying to make functional extracts. Discard any batches of eggs that have "puff balls" or activated eggs that constitute more than 10% of the eggs. Use laid eggs and collect eggs at about 16 to 17 hours after priming with Progesterone (found in Pregnant Mare Serum Gonadotropin (PMSG)). - [Read Cytostatic Factor (CSF) Extract Preparation for Xenopus Spindle Assembly Protocol]

Category: Xenopus Protocols: Xenopus Injection Protocols

Differences in injection of X. laevis and X. tropicalis. Includes: X. tropicalis lays eggs about 4 hours after a boost of hCG; In vitro fertilization is not nearly as efficient in trops compared to laevis; X. tropicalis embryos are much softer than X. laevis embryos; X. tropicalis embryos whose jelly coats are removed by cysteine have a loose sticky vitelline membrane; X. tropicalis do not have a "summer slump". - [Read Differences in Injection of X. laevis and X. tropicalis]

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: PCR Protocols

Method describes how to modify the termini of PCR products by introducing restriction sites and other features.  To reduce the chance of contamination with exogenous DNAs, prepare and use a special set of reagents and solutions for PCR only.  Bake all glassware for 6 hours at 150°C and autoclave all plasticware. - [Read Genetic Engineering with PCR Protocol]

Category: PCR Protocols: PCR Optimization

Method describes how to modify the termini of PCR products by introducing restriction sites and other features.  To reduce the chance of contamination with exogenous DNAs, prepare and use a special set of reagents and solutions for PCR only.  Bake all glassware for 6 hours at 150°C and autoclave all plasticware. - [Read Genetic Engineering with PCR Protocol]

Category: Pharmacology and Toxicology Protocols: Drug Discovery

High-throughput and sensitive assay to measure yeast cell growth: a bench protocol for testing genotoxic agents. Method is highly sensitive, provides quantifiable data and offers high-throughput screening capability.  Starting with the treatment of cells with different doses of damaging agents, pre-prepared growing media containing 96-well plates are inoculated and cell population is automatically monitored every 10 min for 48 hours.  - [Read High Throughput and Sensitive Assay Measure Yeast Cell Growth Protocol]

Category: Immunohistochemistry Protocols

Protocol for indirect peroxidase technique. Includes: Sections: Thickness-Between 5 and 10 microns. Pick up on chromic acid etched, poly-L-lysine or chrome alum/gelatine coated slides. Dry at room temperature overnight (not longer than 72 hours). - [Read Indirect Peroxidase Technique Protocol]

Category: PCR Protocols: Standard PCR Protocol

Protocol can be used to amplify DNA up to 25 kb in length.  To reduce the chance of contamination with exogenous DNAs, prepare and use a special set of reagents and solutions for PCR only.  Bake all glassware for 6 hours at 150°C and autoclave all plasticware. - [Read Long PCR Protocol]

Category: Drosophila Protocols: Drosophila Staining Protocols

Early embryos (0-17 hours or until cuticle formation) are treated with a mixture of organic solvents, formaldehyde, and alcohols, as described here. The cuticles of late-stage embryos are usually opened by sonication. Tissues from more advanced stages of development are normally dissected by hand and then fixed and stained in a standard paraformaldehyde/detergent combination - [Read Preparing Early Whole-Mount Drosophila Embryos for Immunostaining Protocol]

Category: Drosophila Protocols: Drosophila Staining Protocols

Early and late embryos are treated with a mixture of organic solvents, formaldehyde, and alcohols. The cuticles of late-stage embryos (17-22 hours or until hatching) are usually opened by sonication, as described here. Tissues from later stages of development are normally dissected by hand and then fixed and stained in a standard paraformaldehyde/detergent combination. - [Read Preparing Late Whole-Mount Drosophila Embryos for Immunostaining Protocol]

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]

Category: DNA Protocols: cDNA Library Protocols: cDNA Library Construction Protocols

An expression library constructed in a bacteriophage {lambda} vector is plated on an appropriate E. coli strain in the absence of isopropylthio-ß-D-galactoside (IPTG). After 2-4 hours, the plates are moved to 37°C (to stabilize any fusion proteins that are temperature sensitive), and filters impregnated with IPTG are laid on top of the developing plaques. - [Read Screening Expression Libraries Constructed in Bacteriophage Lambda Vectors Protocol]

Category: DNA Protocols: cDNA Library Protocols

A cDNA library constructed in a plasmid expression vector of the pUC, pUR, or pEX series is plated on agar medium and then replicated onto filters, which are transferred to plates containing IPTG. After 2-4 hours of induction, the colonies are lysed with chloroform and then screened with appropriate antibodies. - [Read Screening Expression Libraries Constructed in Plasmid Vectors Protocol]

Category: Stem Cell Protocols: Stem Cell Culture Protocols

Protocol describes how to set up microdrop cultures to produce embryos which can then be used for making chimeras. The microdrop culture should be set up several hours to 1 day before the experiment to permit temperature and gas equilibration. - [Read Setting Up Microdrop Cultures Protocol]

Category: Cytogenetics Protocols: Fluorescent In Situ Hybridization FISH Protocol

Protocol for small marker chromosome identification in metaphase and interphase using centromeric multiplex FISH (CM-FISH). This approach allows the identification of marker chromosomes and other aneuploides on one single cytogenetic slide, in less than two hours. - [Read Small Marker Chromosome Identification in Metaphase and Interphase using Centromeric Multiplex FISH]

Category: Fungi Protocols: Neurospora Protocols

DNA isolation method yields an average of 0.6 micrograms of genomic DNA that is suitable for Southern analysis or PCR. Starting with fresh mycelium, 20 to 40 samples can be processed in approximately two hours. Better yields (about 5 micrograms) may be obtained by suspending approximately 100 microliters of ground lyophilized mycelium in 500 microliters of isolation buffer and following the protocol. - [Read Small Scale DNA Preps for Neurospora crassa Protocol]

Category: Primary Cell Isolation and Culture Protocols

This protocol describes a method for static culture of early postimplantation mouse embryos on a microscope stage. Embryos between 6.5 and 9.5 days post coitum (dpc) can be cultured and imaged for 24 hours, with very little growth retardation. - [Read Static Culture of Postimplantation Embryos for Imaging Protocol]

Category: Mouse Protocols: Mouse Cell Culture Protocols

Protocol describes a method for static culture of early postimplantation mouse embryos on a microscope stage. Embryos between 6.5 and 9.5 days post coitum (dpc) can be cultured and imaged for 24 hours, with very little growth retardation. - [Read Static Culture of Postimplantation Embryos for Imaging Protocol]

Category: Cell Culture Protocols

Protocol describes static culture of postimplantation embryos, an alternative to the roller method. The static method is best suited to 6.0 to 7.0 days post coitum (dpc) embryos followed for 24 hours (7.0 dpc embryos) to 48 hours (6.0 dpc embryos) of development. It allows repetitive real-time observation with minimal handling of the embryo. It is especially useful if single or small groups of embryos need to be distinguished from each other. - [Read Static Culture of Postimplantation Embryos Protocol]

Category: Mouse Protocols: Mouse Embryology Protocols

Blastocyst transfer is usually performed 24 hours after aggregation, when the morulae have become expanded blastocysts, and on the same day as injection. A little time is given between injection and transfer to allow the blastocysts to re-expand. Includes: The Mouse Recipient; The Transfer. - [Read Transfer of Injected Blastocysts to Pseudo Pregnant Mice Protocol]

Category: Protein Protocols: Western Blot Protocol: Troubleshooting Western Blots

Troubleshooting Guide for Chemiluminescent Western Blotting Pierce. High Background, Reverse image on film, Blot glows in the darkroom, Signal duration is less than 8 hours, Weak or no signal, Spots within the protein bands, non-specific bands. - [Read Troubleshooting Guide for Chemiluminescent Western Blotting Pierce]