Genetic Protocols for Pristionchus pacificus. Includes: Freezing worms; EMS mutagenesis; Psoralen mutagenesis; Construction of deletion libraries to generate P. pacificus gene knock-outs; Designing primers for the gene of interest; RNAi and morpholino by injection. - [Read Pristionchus pacificus Genetic Protocols]
Recycle tubulin fractions stored at -80¡C after the PC column and store the recycled tubulin in small aliquots for day-to-day use. Generally store recycled tubulin in Injection Buffer (IB) without free GTP. This is done because depolymerization appears to be much better in IB, IB is ideal for microinjections/adding tubulin to extracts, and the absence of free GTP makes polymerization with GMPCPP, a very useful GTP analog that has ~5-10X lower affinity than GTP for tubulin. - [Read Recycling Tubulin Protocol]
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]
This protocol describes a method for visualizing early embryo implantation sites using Chicago Sky Blue 6B dye. Once implantation and interimplantation sites are identified and separated, they can be used for cellular, biochemical, and molecular biology analyses. - [Read Visualizing Early Embryo Implantation Sites by Dye Injection Protocol]
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.
The protocol gives general considerations for the design of targeting vectors for transgenic mice. The protocol shares tips in the design of knock-out and knock-in vectors and some of their strategies for producing homologously recombined embryonic stem cells.