Sperm dilution buffer

Sperm dilution buffer 250mM sucrose, 75mM KCl, 0.5 mM spermidine trihydrochloride, and 0.2 mM spermine tetrahydrochloride. Add about 80 xL of 0.1N NaOH per 20mL solution to titrate to pH 13-1.5 and store 0.5-1 mL aliquots at -20°C. 

Allow an aliquot of sperm dilution buffer (SDB) to equiUbrate to room temperature, and make up 2.5% cysteine in lx MMR, pH 8.0. 

Count of the sperm nuclei After the final spin, resuspend the peUet in l(X)pL of sperm storage buffer. For a 1 100 dilution of our sperm stock, we typically obtain counts of 80 (xlO nuclei/mL) in 1-mm x 1-mm x 0.1-mm square of an improved Neubauer hemacytometer. At this concentration, the undiluted stock contains 8 x lO nuclei/pL, and we use lOpL of that for a reaction. If the sperm stock is substantially less concentrated, let the sperm settle for a few hours or overnight, and remove some of supernatant. If the count is still low, use up to 30 pL of the sperm nuclei per reaction. Sperm nuclei cau be frozen in aliquots at -80°C. 

Phase-contrast microscopy provides a fast and reliable way to evaluate the onset of sperm chromatin decondensation and the appearance of nuclei assembled in vitro (Fig. 1). After incubation, a IO-/1I aliquot of the incubation mixture should be taken and immediately fixed with an equal volume of 8% (w/v) paraformaldehyde in 154 mM PIPES-NaOH, pH 7.5, for 5 min on ice. After fixation, samples should be diluted with an equal volume of extraction buffer as specified above. Then, 5-ix aliquots of this mixture may then be mounted for viewing between slide and cover slip. To ensure adequate separation between the slide and cover slip, about 2 /xl of melted bee s wax should be deposited onto each corner of the cover slip before mounting. 

To isolate chromatin-associated proteins following incubation of sperm nuclei in Xenopus egg extract, the mixture should first be diluted with buffer XN (Philpott and Leno, 1992 Leno et ai, 1996) and the nuclei pelleted by centrifugation at 3500 rpm for 10 min in an SW50.1 rotor (Beckman). The nuclei are then resuspended in buffer, recentrifuged under the same conditions, and finally... 

Reaction of the sperm nuclei Use lOpL of sperm nuclei (-8 x 1(P nuclei) per reaction. After the incubation with egg extract, dilute all the reaction mix with 130 pL of MOH that has equilibrated at room temperature. Mix well and backfill a needle using a clipped yellow tip with a piece of tube. If you use more than lOpL of the sperm (up to 30 pL), the amount of MOH buffer should be reduced. The concentration of nuclei must be considerably higher with X. tropicalis, since the volume injected into each egg is about one fifth the amount used for X. laevis. 

---