Protocol describes a method for synchronizing monolayer cells in mitosis using selective detachment from their substrate. During mitosis, cells become spherical, causing them to become more loosely attached to their substrate. The "rounded up" cells are selectively detached by tapping the culture flask, resulting in a population in which as many as 90-98% of the cells are in mitosis. The drug nocodazole is used to increase the percentage of cells undergoing mitosis before detachment is performed - [Read Synchronization of Mammalian Cell Cultures in Mitosis Using Selective Detachment Protocol]
This protocol describes a method for synchronizing monolayer cells in mitosis using selective detachment from their substrate. During mitosis, cells become more spherical, causing them to become more loosely attached to their substrate. The "rounded up" cells are selectively detached by tapping the culture flask, resulting in a population in which as many as 90-98% of the cells are in mitosis. The drug nocodazole is used to increase the percentage of cells undergoing mitosis before detachment.. - [Read Synchronization of Mammalian Cell Cultures in Mitosis Using Selective Detachment Protocol]
The basis of this test is that a cytotoxic chemical (regardless of site or mechanism of action) will interfere with the normal motility of the protozoan, Tetrahymena thermophila, in culture. The degree of interference of motility as compared to control cultures, related to the concentration of the test compound, provides an indication of toxicity. - [Read Tetrahymena Thermophila Ocular Irritancy Test]
This protocol describes two transfection methods for expressing GFP-tagged actin in primary neurons. The lipid reagent DOTAP (Roche Diagnostics) method produces actin-GFP-expressing hippocampal neurons that survive well during long periods in culture. The calcium phosphate method can be used to transfect neurons that have already been growing on coverslips in vitro. Transfected cells suitable for imaging can be obtained in cultures up to 15 days in vitro. - [Read Transfecting Cultured Hippocampal Neurons with an Actin-GFP Plasmid]
Electrocompetent bacteria are prepared by growing cultures tomid-log phase, washing the bacteria extensively at low temperature,and then resuspending them in a solution of low ionic strengthcontaining glycerol. DNA is introduced during exposure of thebacteria to a short high-voltage electrical discharge.
Simple method for growing Neurospora and for isolation of DNA that may be performed in two days from start to finish. The growth of mycelia in Petri plates eliminates the need for large numbers of flasks when growing many cultures for DNA isolation. - [Read Ultra-Fast Method of DNA Extraction from Neurospora Protocol]
No cell culture problem is as universal as that of culture loss due to contamination. All cell culture laboratories and cell culture workers have experienced it. Culture contaminants may be biological or chemical, seen or unseen, destructive or seemingly benign, but in all cases they adversely affect
both the use of your cell cultures and the quality of your research. Contamination problems can be divided into three classes: Minor annoyances, Serious problems, Major catastrophes. - [Read Understanding and Managing Cell Culture Contamination Protocol]
This protocol describes the electroporation of the BMH 81-17 mut S strain that is recommended for tranformation of the site directed mutagenesis of dsDNA (See Protocol on Site-Directed Mutagenesis on Double Stranded DNA). BMH 81-17 mut S are a mismatch repair defective (mut S) Escherichia coli strain. The probability that the two mutations will cosegregate during the first round of DNA replication is increased in this strain.