Sucrose Buffers for Preparation of Demembranated Sperm Chromatin

Reagent	Buffer #1 (0.2 m sucrose)	Buffer #2 (2.3 m sucrose)	Buffer #3 (2.5 m sucrose)	Buffer #4 (2.0 m sucrose)	Buffer #5 (0.5 m sucrose)
Buffer X (10×)	10 mL	1 mL	1 mL	1 mL	1 mL
Sucrose	6.84 g	7.87 g	8.56 g	6.84 g	1.71
BSA					300 mg
H₂O	to 100 mL	to 10 mL	to 10 mL	to 10 mL	to 10 mL
For Buffer #6 (0.2 m sucrose), add 300 mg of BSA to 10 mL of Buffer #1. Use BSA from Sigma-Aldrich (A7906). Do not use other types of BSA, as they can ruin the sperm preparation. For all buffers, use ultrapure sucrose (≥99%; Invitrogen 15503-022). For Buffers #5 and #6 and for 5 mL of Buffer #1, include 10 µg/mL aprotinin (Roche 11583794001), 10 µg/mL leupeptin (Roche 11529048001), and 1 mm DTT (Bio-Rad 161-0611). Make all buffers fresh on the day of extract preparation.