This protocol is essentially as described by Murray (1991), Cell Cycle Extracts. In Methods in Cell Biology, B.K. Kay and B. Peng, eds. (San Diego: Academic Press), pp. 581-605. I’ve included a protocol which emphasizes the points that we find are most important for obtaining good CSF extracts that are competent f or CSF spindle assembly and for cycled spindle assembly. The indicated buffer amounts are sufficient for a 4 frog prep. For sperm nucleus preparation, see above protocol. For spindle assembly, see Sawin and Mitchison (1991) J. Cell Biol. 112: 925-940.

　　Notes Before Beginning: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 as more than 10% of the eggs. We routinely use laid eggs and collect at about 16-17 hours. If you are trying to make extracts that will form spindles competent of anaphase chromosome segregation, we find it necessary to use only freshly squeezed eggs. Keep the eggs cool (16 deg C incubator) and only bring them to RT right before you are ready to prepare the extract.

　　Stock Buffers:Extract Prep Buffers:EquipmentBefore Starting

　　Protocol

　　Extract is Ready to go!