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]
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]
There are essentially three parts to this protocol: 1. growth of at least 5x10e8 pfu phage to provide an inoculum growth of a larger liquid lysate that will produce about 5x10e12 pfu; 2. concentration and purification of the phage, and; 3. DNA preparation. - [Read Growth and Purification of 25-100 ug Lambda Clone DNA Protocol]
Protocol was designed to rapidly generate small scale cytosolic extracts of C. elegans for Western or IP (has not been tested for RNA work). The protocol works well for between 50 to 5000 worms and has not been extensively tested on larger a scale, though it should work. Includes: Collection; Sonication; Clearing lysate; Immunoprecipitation. - [Read Preparation of Worm Extracts by Sonication Protocol]
Tissue subcellular fractionation and protein extraction for use in mass-spectrometry-based proteomics. Although many methods exist for fractionating proteins, the method described here can capture the majority of subcellular fractions simultaneously at reasonable purity. The scalability of this method makes it amenable to small samples, such as embryonic tissues, in addition to larger tissues. The protocol described is for the general fractionation and extraction of proteins from organs / tissue - [Read Tissue subcellular fractionation and protein extraction for use in mass-spectrometry-based proteomic]
DNA microarrays are an ordered arrangement of DNA molecules complementary to genes of interest that are "spotted" by robotic equipment onto a glass slide substrate. The expression of genes in cells can be monitored with microarrays by preparing cDNA from the mRNA of cells of interest and measuring the hybridization to the microarray. This protocol describes the labeling of genomic DNA for use as a probe for hybridization to the cDNA spotted on the array.
This protocol a protocol on how to generate transfected embryonic stem (ES) cell clones. The previous protocol in this series is the Protocol for Electroporation of ES cells. The next protocol in the series is the Protocol on Disaggregation, Expansion, and Freezing of Transfected ES Clones.