Protocol describes a method for assembling aggregates between ES cells and diploid embryos. The resulting chimeras are useful for separating certain extraembryonic phenotypes from phenotypes in the embryo proper, since the diploid embryo contributes to all parts of the conceptus, but the ES cell component does not contribute to the trophoblast or yolk sac endoderm. - [Read Assembling Aggregates between Embryonic Stem (ES) Cells and Diploid Embryos Protocol]
Cryogenic preservation (storage below -100°C) of cell cultures is widely used to maintain backups or reserves of cells without the associated effort and expense of feeding and caring for them. The success of the freezing process depends on four critical areas: Proper handling and gentle harvesting of the cultures; Correct use of the cryoprotective agent; A controlled rate of freezing; Storage under proper cryogenic conditions. - [Read Cryogenic Preservation and Storage of Animal Cells Protocol]
Protocol for the dissection of Drosophila CNSs. Includes: Tools; Dissection of the CNS; Dissecting larval CNSs; Dissecting early pupae; Dissecting mid pupae; Dissecting late pupae; Dissecting the whole CNS; Dissecting only the brain proper; Taking the pupa out of the puparium. - [Read Dissection of Drosophila CNSs Protocol]
This protocol describes the storage of "homemade" purified antibodies. Antibodies from commercial sources are normally supplied with proper storage conditions. - [Read Storage of Purified Antibodies]
A Single Stranded Plasmid DNA Isolation Protocol describing the production and isolation of single-stranded DNA (ssDNA) using bacteriophagemid-containing bacteria and helper phage. Infection of the host cells with helper phage allows for packaging of ssDNA into bacteriophage. The ssDNA can then be isolated from phage particles.
Tubulin is polymerized into microtubules by incubating tubulin at 37°C with GTP. A nucleation seed is added when the purpose is to assay microtubule elongation. Tubulin can also be polymerized for the purposes of recycling the tubulin or labeling the microtubules with fluorescently labeled tubulin. Based on the protocol by Timothy Mitchison of Harvard University.
A single step RNA isolation protocol using Phenol Chloroform Extraction and Acid Guanidinium Thiocyanate. This RNA isolation method uses the fact that guanidinium thiocyanate can simultaneously lyse the cells and inactive cellular RNAses during the initial RNA isolation step allow a single step in the method.
In Vitro Translated Xenopus Mos Kinase Assay Protocol. In response to progesterone, immature Xenopus oocytes mature to eggs that can be fertilized. The Mos protein kinase is essential for oocyte maturation, most likely due to its ability to activate the MAP kinase cascade. This MAP kinase cascade eventually leads to the activation of Cdc2/cyclin B and entry into M phase. In this protocol, tagged Mos kinase is translated in vitro, immunopurified, and used in a kinase assay.
3' Rapid Amplification of cDNA Ends RACE Using PCR Protocol. This protocol contains the steps for 3' end rapid amplification of mRNA by PCR. The first-strand cDNA is synthesized from total or poly(A+) RNA by priming from the poly-A tail of the mRNA using a oligo (dT) adaptor primer. The cDNA is then amplified via PCR using a gene-specific primer and an adaptor primer.
Histone H1 Kinase Activity Assay Protocol. This protocol describes assaying kinase activity of a putative kinase using Histone H1 as the substrate. Histone H1 is the canonical kinase substrate in this type of assay. Phosphorylation of Histone H1 is assessed by SDS-Polyacrylamide gel electrophoresis followed by autoradiography.